SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy.

BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion.

Interleukin-1ß (IL-1ß)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1ß production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1ß expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1ß (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8+ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy.

Novel Pathophysiological, Diagnostic and Therapeutic Concepts in Acute and Recurrent Pericarditis.

Acute pericarditis is the most frequent pericardial disease characterized by inflammation of the pericardial layers resulting in pain, dyspnea and fatigue. Often limited to an isolated event, up to 30% of patients experience one or more recurrences. There is limited knowledge about the pathophysiology of this disease, possibly due to the limited availability of animal models. More recently, following seminal clinical trials with colchicine and interleukin-1 (IL-1) blockers and a novel murine model of acute pericarditis using zymosan A, it has become clear that the NLRP3 (NACHT, leucine-rich repeat, and pyrin domain-containing protein 3) inflammasome/IL-1 ß axis plays a central role in driving acute pericardial inflammation and in sustaining this process during recurrences. Diagnostic management of pericarditis has been implemented with multimodality imaging including echocardiography, cardiac computed tomography, and cardiac magnetic resonance. These imaging modalities provide essential diagnostic and pathogenetic information, and are able to characterize pericardial inflammation, allowing to refine risk stratification and personalize treatment. Recent acquisitions yield relevant implications with regard to the therapeutic management of acute and recurrent pericarditis. Non-steroidal anti-inflammatory drugs (NSAIDs) and colchicine are cornerstone therapies either for acute and recurrent pericarditis. However, the benefits of targeted agents, such as anakinra - a recombinant human IL-1 receptor antagonist - and rilonacept - an IL-1 α /IL-1 ß trap, are being increasingly recognized. To this end, phenotyping patients with pericarditis and addressing such therapies to those presenting with auto-inflammatory features (elevated C-reactive protein, sustained pericardial and systemic inflammation, multiple recurrences) is of utmost importance to identify patients who might be more likely to benefit from NLRP3 inflammasome/IL-1 ß pathway blockade.

The interleukin-1 receptor type I promotes the development of aging-associated cardiomyopathy in mice.

BACKGROUND: Aging is associated with metabolic and structural changes causing heart failure with preserved ejection fraction (HFpEF). Interleukin-1 (IL-1) is a pro-inflammatory cytokine involved in aging-related inflammation. OBJECTIVE: We sought to determine whether IL-1 mediates aging-related changes in the heart, as seen in HFpEF. METHODS: We studied age-matched young (4-month-old), middle-aged (14-month-old), and old (23-month-old) wild-type (WT) C57BL/6J and IL-1 receptor type I deficient (IL1RI-KO) male mice. Echocardiography was used to evaluate left ventricular (LV) dimensions and systolic/diastolic function, and a pressure transducer was used to measure the LV end-diastolic pressure. Picrosirius red stain was used to assess for myocardial interstitial fibrosis (MIF) at pathology. RESULTS: WT and IL-1RIKO mice showed a normal cardiac phenotype at young age, without any differences between the two groups. With aging, the WT mice developed LV concentric hypertrophy (as measured by a significant increase in LV mass [+42%, P < 0.01] and relative wall thickness [+34%, P < 0.01]), whereas the aging IL-1RI-KO mice did not. With aging, the WT mice also developed diastolic dysfunction (as measured by a significant increase in isovolumetric relaxation time [+148%, P < 0.01] and a significantly higher LV end-diastolic pressure [+174%, P < 0.01]), whereas the aging IL1RI-KO did not. Aged WT mice showed a significant increase in MIF (+124%, P < 0.01) at cardiac pathology, whereas the aging IL-1RI-KO did not. CONCLUSIONS: Genetically-modified mice lacking the IL-1RI receptor, not responsive to IL-1, are protected from aging-related LV hypertrophy, fibrosis, and diastolic dysfunction. These data support a central role of IL-1 in the pathophysiology of aging-related HFpEF.

Assessment of machine perfusion conditions for the donation after circulatory death heart preservation.

BACKGROUND: Donation after circulatory death (DCD) hearts requires machine perfusion preservation, the conditions of which are not well defined. METHODS: To achieve this, rat hearts were procured following a DCD or control beating-heart donation (CBD) model, and perfused for 60 min with one of three machine perfusion solutions-St. Thomas (ST), University of Wisconsin (UW), or Polyethylene Glycol-20k (PEG)-at one of two temperatures, 4°C or 15°C. At 15-min intervals, perfusion pressure was measured as a marker of vascular resistance. Colored microspheres were added to capture the distribution of perfusate into the metabolically active sub-endocardium, and the eluate was collected for troponin assays. Analyses compared groups using Wilcoxon rank-sum and ANOVA. RESULTS: Perfusion pressure was significantly higher for DCD than CBD hearts at 15°C regardless of solutions. The lowest rise in perfusion pressure over time was observed with PEG at 15°C. Except for PEG at 15°C, ST and UW solutions at 4 or 15°C had decreased sub-endocardial perfusion in DCD hearts. Troponin release from DCD hearts with UW and PEG solutions was comparable to CBD hearts but was significantly higher with ST solution at 15°C. CONCLUSIONS: Optimal preservation conditions for DCD hearts were observed with PEG machine perfusion solution at 15°C.

Inflammasome formation in the lungs of patients with fatal COVID-19.

OBJECTIVE: The orf8b protein of the coronavirus SARS-CoV, analogous to SARS-CoV-2, triggers the NLRP3 inflammasome in macrophages in vitro. Deregulated inflammasome-mediated release of interleukin-1 family cytokines is important in hyper-inflammatory syndromes, like happens in SARS-CoV-2-mediated cytokine release syndrome. We propose that an intense inflammasome formation characterizes the lungs of patients with fatal COVID-19 disease due to pneumonia and acute respiratory distress syndrome (ARDS). METHODS: Samples from four patients with confirmed COVID-19 pneumonia who had been hospitalized at the Hospital of the University of Trieste (Italy) and died of ARDS and four lung samples from a historical repository from subjects who had died of cardiopulmonary arrest and had not been placed on mechanical ventilation and without evidence of pulmonary infection at postmortem examination were collected. Pathology samples had been fixed in formalin 10% at time of collection and subsequently embedded in paraffin. We conducted staining for ASC (Apoptosis-associated Speck-like protein containing a Caspase recruitment domain), NLRP3 (NACHT, LRR, and PYD domains-containing protein 3), and cleaved caspase-1. RESULTS: Intense expression of the inflammasome was detected, mainly in leukocytes, within the lungs of all patients with fatal COVID-19 in the areas of lung injury. The number of ASC inflammasome specks per high power fields was significantly higher in the lungs of patients with fatal COVID-19 as compared with the lungs of control subjects (52 ± 22 vs 6 ± 3, P = 0.0064). CONCLUSIONS: These findings identify the presence of NLRP3 inflammasome aggregates in the lungs of fatal COVID-19 pneumonia thus providing the potential molecular link between viral infection and cytokine release syndrome.

Preservation of Contractile Reserve and Diastolic Function by Inhibiting the NLRP3 Inflammasome with OLT1177® (Dapansutrile) in a Mouse Model of Severe Ischemic Cardiomyopathy Due to Non-Reperfused Anterior Wall Myocardial Infarction.

Interleukin-1ß (IL-1ß), a product of the NLRP3 inflammasome, modulates cardiac contractility and diastolic function. We proposed that OLT1177® (dapansutrile), a novel NLRP3 inhibitor, could preserve contractile reserve and diastolic function after myocardial infarction (MI). We used an experimental murine model of severe ischemic cardiomyopathy through the ligation of the left coronary artery without reperfusion, and after 7 days randomly assigned mice showing large anterior MI (>4 akinetic segments), increased left ventricular (LV) dimensions ([LVEDD] > 4.4 mm), and reduced function (LV ejection fraction < 40%) to a diet that was enriched with OLT1177® admixed with the chow in the diet at 3.75 g/kg (Group 1 [n = 10]) or 7.5 g/kg (Group 2 [n = 9]), or a standard diet as the no-treatment control group (Group 3 [n = 10]) for 9 weeks. We measured the cardiac function and contractile reserve with an isoproterenol challenge, and the diastolic function with cardiac catheterization at 10 weeks following the MI surgery. When compared with the control (Group 3), the mice treated with OLT1177 (Group 1 and 2) showed significantly greater preservation of their contractile reserve (the percent increase in the left ventricular ejection fraction [LVEF] after the isoproterenol challenge was +33 ± 11% and +40 ± 6% vs. +9 ± 7% in the standard diet; p < 0.05 and p < 0.005 for Group 1 and 2, respectively) and of diastolic function measured as the lower left ventricular end-diastolic pressure (3.2 ± 0.5 mmHg or 4.5 ± 0.5 mmHg vs. 10.0 ± 1.6 mmHg; p < 0.005 and p < 0.009 respectively). No differences were noted between the resting LVEF of the MI groups. These effects were independent of the effects on the ventricular remodeling after MI. NLRP3 inflammasome inhibition with OLT1177® can preserve ß-adrenergic responsiveness and prevent left ventricular diastolic dysfunction in a large non-reperfused anterior MI mouse model. OLT1177® could therefore be used to prevent the development of heart failure in patients with ischemic cardiomyopathy.

NLRP3 Inflammasome in Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is associated with the induction of a sterile inflammatory response that leads to further injury. The NACHT, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a macromolecular structure responsible for the inflammatory response to injury or infection. NLRP3 can sense intracellular danger signals, such as ischemia and extracellular or intracellular alarmins during tissue injury. The NLRP3 inflammasome is primed and triggered by locally released damage-associated molecular patterns and amplifies the inflammatory response and cell death through caspase-1 activation. Here, we examine the scientific evidence supporting a role for NLRP3 in AMI and the available strategies to inhibit the effects of the inflammasome. Our focus is on the beneficial effects seen in experimental models of AMI in preclinical animal models and the initial results of clinical trials.

The NLRP3 Inflammasome Inhibitor, OLT1177 (Dapansutrile), Reduces Infarct Size and Preserves Contractile Function After Ischemia Reperfusion Injury in the Mouse.

BACKGROUND: Activation of the NLRP3 inflammasome is a primary driver of sterile inflammation in response to myocardial ischemia reperfusion. Pharmacologic inhibitors of the NLRP3 inflammasome are being developed. We proposed that OLT1177 (dapansutrile), a novel NLRP3 inflammasome inhibitor, could preserve myocardial function after ischemia reperfusion injury in the mouse. METHODS: We used an experimental murine model of myocardial ischemia reperfusion injury through transient ligation of the left coronary artery and measured the effects of OLT1177 (6, 60, or 600 mg/kg intraperitoneal dose) on infarct size at pathology and on systolic cardiac function at echocardiography. To simulate a clinical scenario, we investigated the time window of therapeutic intervention with OLT1177 (60 mg/kg) administered 60, 120, or 180 minutes after reperfusion. RESULTS: OLT1177 was rapidly detectable in the plasma following intraperitoneal injection and had no effect on cardiac function in healthy mice. OLT1177 treatment at reperfusion showed significant dose-dependent reduction in infarct size (-36%, -67%, and -62% for 6, 60, and 600 mg/kg, respectively; P < 0.001 for linear trend, P = 0.010 vs. vehicle for 6 mg/kg, and P < 0.001 vs. vehicle for 60 and 600 mg/kg) and preserved cardiac systolic function measured as left ventricular fractional shortening at 24 hours and 7 days after injury (P = 0.015 for 6 mg/kg and P < 0.01 for 60 and 600 mg/kg). OLT1177 reduced infarct size also when given after 60 minutes of reperfusion (-71%, P < 0.001 vs. vehicle). CONCLUSION: OLT1177 (dapansutrile) limits infarct size and prevents left ventricular systolic dysfunction when given within 60 minutes following ischemia reperfusion injury in the mouse.

Inflammasome, pyroptosis, and cytokines in myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury induces a sterile inflammatory response, leading to further injury that contributes to the final infarct size. Locally released danger-associated molecular patterns lead to priming and triggering of the NOD-like receptor protein 3 inflammasome and amplification of the inflammatory response and cell death by activation of caspase-1. We review strategies inhibiting priming, triggering, or caspase-1 activity or blockade of the inflammasome-related cytokines interleukin-1ß and interleukin-18, focusing on the beneficial effects in experimental models of acute myocardial infarction in animals and the initial results of clinical translational research trials.

Inhibiting the Inflammatory Injury After Myocardial Ischemia Reperfusion With Plasma-Derived Alpha-1 Antitrypsin: A Post Hoc Analysis of the VCU-α1RT Study.

BACKGROUND: Despite the benefits of reperfusion in limiting myocardial injury, the infarct size continues to expand after reperfusion because of secondary inflammatory injury. Plasma-derived alpha-1 antitrypsin (AAT) inhibits the inflammatory injury in myocardial ischemia and reperfusion. To explore the effects of plasma-derived AAT on the inflammatory response to ischemia-reperfusion injury, we analyzed time-to-reperfusion and enzymatic infarct size estimates in a post hoc analysis of the VCU-α1RT clinical trial (clinicaltrials.gov NCT01936896). METHODS: Ten patients with ST-segment elevation acute myocardial infarction (STEMI) were enrolled in an open-label, single-arm treatment study of Prolastin C, plasma-derived AAT, at 60 mg/kg infused intravenously within 12 hours of reperfusion. Biomarkers were measured serially over the first 72 hours, and patients were followed clinically for the occurrence of new-onset heart failure, recurrent MI, or death. Twenty patients with STEMI who had been enrolled in previous randomized trials with identical inclusion/exclusion criteria and had been assigned to placebo served as historical controls. RESULTS: Time to percutaneous coronary intervention and time to drug did not significantly differ between groups. AAT-treated patients had a significantly shorter time-to-peak creatine kinase myocardial band (CK-MB) values (525 [480-735] vs. 789 [664-959] minute, P = 0.005) and CK-MB area under the curve (from 1204 [758-2728] vs. 2418 [1551-4289] U·day, P = 0.035), despite no differences in peak CK-MB (123 [30-196] vs. 123 [71-213] U/mL, P = 0.71). CONCLUSIONS: A single administration of Prolastin C given hours after reperfusion in patients with STEMI led to a significant shorter time to peak and area under the curve for CK-MB, despite similar peak CK-MB values. These preliminary data support the hypothesis that Prolastin C shortens the duration of the ischemia-reperfusion injury in patients with STEMI.

An Orally Available NLRP3 Inflammasome Inhibitor Prevents Western Diet-Induced Cardiac Dysfunction in Mice.

BACKGROUND: A diet rich in saturated fat and sugars (Western diet, WD) induces myocardial expression of the NLRP3 inflammasome and dysfunction in mice. We therefore hypothesized that a diet enriched with an orally available NLRP3 inflammasome inhibitor could prevent WD-induced cardiac dysfunction in mice. METHODS: Ten-week-old CD-1 male mice were fed WD or standard diet (SD) for 8 weeks. The compound 16673-34-0, an orally active NLRP3 inhibitor, was added to the diet at a concentration of 100 mg/Kg. The plasmatic levels of the NLRP3 inflammasome inhibitor were measured. Food intake, body weight, and glucose tolerance were assessed. Cardiac systolic and diastolic functions were measured by Doppler echocardiography at baseline, 4 weeks, and 8 weeks. RESULTS: WD induced a significant increase in body weight (+14%, P = 0.02), impaired glucose tolerance (+34%, P = 0.03), and a significant increase in isovolumetric relaxation time (+129%, P = 0.03) and reduction in left ventricular ejection fraction (-10%, P = 0.03), as compared to standard chow diet (SD). The treatment with NLRP3 inhibitor in the diet prevented cardiac systolic and diastolic dysfunction (P < 0.05 for left ventricular ejection fraction, isovolumetric relaxation time, and myocardial performance index in WD with drug vs. WD without drug), without significant changes in heart rate and metabolic parameters. CONCLUSIONS: An orally available NLRP3 inhibitor prevented WD-induced cardiac dysfunction in obese mice.

Reduction of Myocardial Ischemia-Reperfusion Injury by Inhibiting Interleukin-1 Alpha.

BACKGROUND: Interleukin-1α (IL-1α) released by dying cells is an alarmin that activates the innate immunity. We hypothesized that after myocardial ischemia-reperfusion (I/R) injury, IL-1α amplifies the myocardial damage by activating the inflammasome and caspase-1. METHODS: Adult male CD1 mice were used. The left anterior descending coronary artery was ligated for 30 minutes, after 24 hours of reperfusion. An IL-1α blocking antibody (15 µg/kg intraperitoneally) or matching vehicle was given after reperfusion. A subgroup of mice underwent sham surgery. We assessed the effects of IL-1α blockade on caspase-1 activity, infarct size, cardiac troponin I serum levels, and left ventricular fractional shortening, 24 hours after I/R. RESULTS: I/R led to inflammasome formation, and IL-1α blockade significantly reduced inflammasome formation, reflected by a >50% reduction in caspase-1 activity versus vehicle (P = 0.03). IL-1α blockade also reduced the infarct size (-52% infarct expressed as percentage of area at risk, and -79% for cardiac troponin I serum levels, P < 0.001 vs. vehicle) and preserved the left ventricular fractional shortening (31 ± 3% vs. 25 ± 2%, P < 0.001 vs. vehicle). CONCLUSION: IL-1α blockade after I/R reduces the inflammasome activation, decreases the infarct size, and preserves the left ventricular function. IL-1α blockade may therefore represent a novel therapeutic strategy to reduce I/R injury.

A Preclinical Translational Study of the Cardioprotective Effects of Plasma-Derived Alpha-1 Anti-trypsin in Acute Myocardial Infarction.

BACKGROUND: The area of myocardial infarction continues to expand for hours after reperfusion. The injured but viable myocardium may be salvaged if the signals leading to cell death are interrupted. Activation of the caspase-1 inflammasome in the heart shortly after ischemia-reperfusion contributes to the final infarct size. Plasma-derived α-1 anti-trypsin (AAT) has shown to inhibit inflammasome formation in vitro and in vivo. To explore the potential translational clinical value of AAT as a therapeutic, we conducted a series of preclinical experiments designed to simulate clinically relevant scenarios. METHODS: Adult male CD1 mice were used. The left anterior descending coronary artery was ligated for 30 or 75 minutes followed by reperfusion, to explore different severity of ischemic injury. Plasma-derived AAT (Prolastin C) was administered intraperitoneally after reperfusion, without pretreatment, exploring 3 different doses (60, 120, and 180 mg/kg). In a subgroup of mice, we administered Prolastin C with a delay of 30 minutes after reperfusion to simulate the clinical context of delayed administration, and we also used a model of permanent coronary artery ligation without reperfusion. Finally, we tested whether a single dose at reperfusion was sufficient to maintain a benefit in the longer term (7 days). Infarct size was measured by 3 different and independent methodologies: pathology, plasma levels of troponin I, and wall motion abnormalities at echocardiography. RESULTS: Prolastin C given at reperfusion after 30 minutes of ischemia provided a powerful reduction in infarct size (>50% reduction in all methodology used, all P < 0.01) without a clear dose-dependent response. Prolongation of ischemia to 75 minutes nor a delay in treatment by 30 minutes after reperfusion had any negative impact on Prolastin C effects. A single dose given at reperfusion was as effective as multiple daily doses. When given to the mouse without reperfusion, Prolastin C failed to reduce infarct size. CONCLUSIONS: Plasma-derived AAT (Prolastin C) given as an adjunct to reperfusion powerfully limits the final infarct size across a wide range of experiments in the mouse reproducing clinically relevant scenarios, such as variable duration of ischemia, delay in administration in the drug, and a large therapeutic index.

Recombinant Human Alpha-1 Antitrypsin-Fc Fusion Protein Reduces Mouse Myocardial Inflammatory Injury After Ischemia-Reperfusion Independent of Elastase Inhibition.

BACKGROUND: Alpha-1-antitrypsin (AAT) is an abundant plasma protein with neutrophil elastase-inhibiting activity, and AAT is available as a plasma-derived therapeutic (pAAT). In experimental myocardial infarction, pAAT reduced acute inflammatory injury because of ischemia-reperfusion. The aim of the present study was to assess the properties of a recombinant protein composed of human AAT fused to the human immunoglobulin (Ig) G1 Fc fragment (rhAAT-Fc) in experimental myocardial infarction. METHODS: Ten-week-old CD1 male mice underwent transient occlusion (30 minutes) of the left anterior coronary artery. rhAAT-Fc (2 mg/kg) or pAAT (60 mg/kg) were administered upon reperfusion. We used human plasma-derived Ig (2 mg/kg) or a matching volume of NaCl 0.9% as control solutions. After 24 hours, infarct size and caspase-1 activity were quantified. The left ventricular ejection fraction (LVEF) was measured by echocardiography at 24 hours and 7 days. A variant of rhAAT-Fc lacking elastase inhibition activity, rhAAT-Fc, was also tested. RESULTS: The rhAAT-Fc induced a significant reduction in infarct size (P < 0.01 vs. all controls, P > 0.05 vs. pAAT). Caspase-1 activity was reduced to the same degree with rhAAT-Fc and pAAT (-70%; P < 0.05; P > 0.05 rhAAT-Fc vs. pAAT). The effects on infarct size after a single administration were reflected by preservation of LVEF at 24 hours and 7 days (all P < 0.05). rhAAT-Fc without elastase inhibiting activity, rhAAT-Fc, conferred comparable effects on infarct size, caspase-1 activity, and LVEF (P > 0.2 vs. rhAAT-Fc). CONCLUSIONS: The pAAT and recombinant human AAT-Fc reduce the acute myocardial inflammatory injury after ischemia-reperfusion in the mouse leading to preservation of viable myocardium and systolic function, independent on the effects on neutrophil elastase.

Role of Interleukin-1 in Radiation-Induced Cardiomyopathy.

Thoracic X-ray therapy (XRT), used in cancer treatment, is associated with increased risk of heart failure. XRT-mediated injury to the heart induces an inflammatory response leading to cardiomyopathy. The aim of this study was to determine the role of interleukin (IL)-1 in response to XRT injury to the heart and on the cardiomyopathy development in the mouse. Female mice with genetic deletion of the IL-1 receptor type I (IL-1R1 knockout mice [IL-1R1 KO]) and treatment with recombinant human IL-1 receptor antagonist anakinra, 10 mg/kg twice daily for 7 d, were used as independent approaches to determine the role of IL-1. Wild-type (wt) or IL-1R1 KO mice were treated with a single session of XRT (20 or 14 gray [Gy]). Echocardiography (before and after isoproterenol challenge) and left ventricular (LV) catheterization were performed to evaluate changes in LV dimensions and function. Masson's trichrome was used to assess myocardial fibrosis and pericardial thickening. After 20 Gy, the contractile reserve was impaired in wt mice at d 3, and the LV ejection fraction (EF) was reduced after 4 months when compared with sham-XRT. IL-1R1 KO mice had preserved contractile reserve at 3 d and 4 months and LVEF at 4 months after XRT. Anakinra treatment for 1 d before and 7 d after XRT prevented the impairment in contractile reserve. A significant increase in LV end-diastolic pressure, associated with increased myocardial interstitial fibrosis and pericardial thickening, was observed in wt mice, as well as in IL-1R1 KO-or anakinra-treated mice. In conclusion, induction of IL-1 by XRT mediates the development of some, such as the contractile impairment, but not all aspects of the XRT-induced cardiomyopathy, such as myocardial fibrosis or pericardial thickening.

A mouse model of heart failure with preserved ejection fraction due to chronic infusion of a low subpressor dose of angiotensin II.

Heart failure (HF) with preserved ejection fraction (HFpEF) is a clinical syndrome of HF symptoms associated with impaired diastolic function. Although it represents ∼50% of patients with HF, the mechanisms of disease are poorly understood, and therapies are generally ineffective in reducing HF progression. Animal models of HFpEF not due to pressure or volume overload are lacking, therefore limiting in-depth understanding of the pathophysiological mechanisms and the development of novel therapies. We hypothesize that a continuous infusion of low-dose angiotensin II (ATII) is sufficient to induce left ventricular (LV) diastolic dysfunction and HFpEF, without increasing blood pressure or inducing LV hypertrophy or dilatation. Osmotic pumps were implanted subcutaneously in 8-wk-old male mice assigned to the ATII (0.2 mg·kg(-1)·day(-1)) or volume-matched vehicle (N = 8/group) for 4 wk. We measured systolic and diastolic arterial blood pressures through a tail-cuff transducer, LV dimensions and ejection fraction through echocardiography, and LV relaxation through pulsed-wave Doppler and LV catheterization. Myocardial fibrosis and cardiomyocyte cross-sectional area were measured. ATII infusion had no effects on systemic arterial blood pressure. ATII induced significant impairment in LV diastolic function, as measured by an increase (worsening) in LV isovolumetric relaxation time, myocardial performance index, isovolumetric relaxation time constant, and LV end-diastolic pressure without altering LV dimensions, mass, or ejection fraction. Chronic infusion of low-dose ATII recapitulates the HFpEF phenotype in the mouse, without increasing systemic arterial blood pressure. This mouse model may provide insight into the mechanisms of HFpEF.

Pharmacologic Inhibition of the NLRP3 Inflammasome Preserves Cardiac Function After Ischemic and Nonischemic Injury in the Mouse.

BACKGROUND: Sterile inflammation resulting from myocardial injury activates the NLRP3 inflammasome and amplifies the inflammatory response mediating further damage. METHODS: We used 2 experimental models of ischemic injury (acute myocardial infarction [AMI] with and without reperfusion) and a model of nonischemic injury due to doxorubicin 10 mg/kg to determine whether the NLRP3 inflammasome preserved cardiac function after injury. RESULTS: Treatment with the NLRP3 inflammasome inhibitor in the reperfused AMI model caused a significant reduction in infarct size measured at pathology or as serum cardiac troponin I level (-56% and -82%, respectively, both P < 0.001) and preserved left ventricular fractional shortening (LVFS, 31 ± 2 vs. vehicle 26% ± 1%, P = 0.003). In the non-reperfused AMI model, treatment with the NLRP3 inhibitor significantly limited LV systolic dysfunction at 7 days (LVFS of 20 ± 2 vs. 14% ± 1%, P = 0.002), without a significant effect on infarct size. In the doxorubicin model, a significant increase in myocardial interstitial fibrosis and a decline in systolic function were seen in vehicle-treated mice, whereas treatment with the NLRP3 inhibitor significantly reduced fibrosis (-80%, P = 0.001) and preserved systolic function (LVFS 35 ± 2 vs. vehicle 27% ± 2%, P = 0.017). CONCLUSIONS: Pharmacological inhibition of the NLRP3 inflammasome limits cell death and LV systolic dysfunction after ischemic and nonischemic injury in the mouse.

Identifying Inflammasome Activation in Human Tissues.

The NACHT, LRR, and PYD domain-containing protein-3 (NLRP3) inflammasome activation is part of a stereotyped cellular response to injury or infection. The NLRP3 inflammasome activation promotes cellular dysfunction and death, leading to local and systemic inflammation, organ dysfunction, and adverse outcome. Immunohistochemistry and immunofluorescence can be used to determine whether the NLRP3 inflammasome components are present in human biopsy or autopsy tissue samples.