Cardiac Macrophages Research Articles

Cardiac macrophages and emerging roles for their metabolism after myocardial infarction.

Interest in cardioimmunology has reached new heights as the experimental cardiology field works to tap the unrealized potential of immunotherapy for clinical care. Within this space is the cardiac macrophage, a key modulator of cardiac function in health and disease. After a myocardial infarction, myeloid macrophages both protect and harm the heart. To varying degrees, such outcomes are a function of myeloid ontogeny and heterogeneity, as well as functional cellular plasticity. Diversity is further shaped by the extracellular milieu, which fluctuates considerably after coronary occlusion. Ischemic limitation of nutrients constrains the metabolic potential of immune cells, and accumulating evidence supports a paradigm whereby macrophage metabolism is coupled to divergent inflammatory consequences, although experimental evidence for this in the heart is just emerging. Herein we examine the heterogeneous cardiac macrophage response following ischemic injury, with a focus on integrating putative contributions of immunometabolism and implications for therapeutically relevant cardiac injury versus cardiac repair.

Macrophage CAPN4 regulates CVB3-induced cardiac inflammation and injury by promoting NLRP3 inflammasome activation and phenotypic transformation to the inflammatory subtype

Exploring the immune mechanism of coxsackievirus B3 (CVB3)-induced myocarditis may provide a promising therapeutic strategy. Here, we investigated the regulatory role of macrophage CAPN4 in the phenotypic transformation of macrophages and NOD-like receptor protein 3 (NLRP3) inflammasome activation. We found that CAPN4 was the most upregulated subtype of the calpain family in CVB3-infected bone marrow-derived macrophages (BMDMs) and Raw 264.7 cells after CVB3 infection and was upregulated in cardiac macrophages from CVB3-infected mice. Conditional knockout of CAPN4 (CAPN4flox/flox; LYZ2-Cre, CAPN4-cKO mice) ameliorated inflammation and myocardial injury and improved cardiac function and survival after CVB3 infection. Enrichment analysis revealed that macrophage differentiation and the interleukin signaling pathway were the most predominant biological processes in macrophages after CVB3 infection. We further found that CVB3 infection and the overexpression of CAPN4 promoted macrophage M1 polarization and NLRP3 inflammasome activation, while CAPN4 knockdown reversed these changes. Correspondingly, CAPN4-cKO alleviated CVB3-induced M1 macrophage transformation and NLRP3 expression and moderately increased M2 transformation in vivo. The culture supernatant of CAPN4-overexpressing or CVB3-infected macrophages impaired cardiac fibroblast function and viability. Moreover, macrophage CAPN4 could upregulate C/EBP-homologous protein (chop) expression, which increased proinflammatory cytokine release by activating the phosphorylation of transducer of activator of transcription 1 (STAT1) and 3 (STAT3). Overall, these results suggest that CAPN4 increases M1-type and inhibits M2-type macrophage polarization through the chop-STAT1/STAT3 signaling pathway to mediate CVB3-induced myocardial inflammation and injury. CAPN4 may be a novel target for viral myocarditis treatment.

Abstract 034: ROCK2 Specific Inhibition Decreases Th17/Treg Balance And Reduces DOCA-salt Hypertension-associated End-Organ Damage

Hypertension is characterized by immune activation and inflammatory end-organ damage, yet there are no therapies for hypertension that specifically target the immune system. We previously demonstrated a critical role for pro-inflammatory CD4+ T cell derived cytokines, interleukin 17A (IL-17A) and IL-21, in the full development of hypertension and hypertension-associated renal and vascular damage. Rho-associated coiled-coil containing protein kinase 2 (ROCK2) has been shown to function as a molecular switch upregulating Th17 and inhibiting anti-inflammatory regulatory T cell (Treg) differentiation in autoimmune diseases. We found that ROCK2 expression is upregulated in splenic and renal CD4+ T cells in a DOCA (deoxycorticosterone acetate)-salt model of hypertension. We hypothesized that hypertensive stimuli increase T cell ROCK2 activity, leading to increased Th17/Treg ratios, and that selective ROCK2 inhibition is a potential novel therapeutic target for hypertension-associated end-organ damage. We showed in vitro that KD025, an orally bioavailable ROCK2 inhibitor, inhibits Th17 proliferation and IL-17A/IL-21 production. We then tested ROCK2 inhibition in vivo where DOCA salt-induced hypertension (uninephrectomy + 100mg DOCA pellet + 1% NaCl drinking water) was initiated in all mice allowing for hypertension to develop for 10 days, followed by treatment with KD025 (50mg/kg daily, n=11) or vehicle (n=8) starting on day 11 through the remainder of the protocol. KD025 treatment significantly attenuated cardiac hypertrophy (8.7±0.3 vs 7.6±0.2 mg/grams body weight, p=0.005) and left ventricular (LV) fibrosis (4.9±1.7 vs 2.4±1.3% LV area, p=0.008). Flow cytometric analysis revealed that KD025 decreased F4/80+ infiltrating cardiac macrophages (24,000±578 vs 18,918±1,430 cells, p=0.011) and decreased the Th17/Treg ratio (0.251±0.041 vs 0.117±0.009, p=0.003) in the heart. Furthermore, KD025 attenuated renal infiltration of CD3+ T cells (59.0±5.6 vs 43.5±3.8 cells/mg tissue, p=0.019) and albuminuria (163.4±24.1 vs 103.1±21.9 ug albumin/mg creatinine, p=0.011). These data indicate that the ROCK2-specific inhibitor KD025 may be a novel therapeutic for the treatment of hypertension and the associated end-organ damage.

SIRT3 Regulates Clearance of Apoptotic Cardiomyocytes by Deacetylating Frataxin.

Efferocytosis is an activity of macrophages that is pivotal for the resolution of inflammation in hypertension. The precise mechanism by which macrophages coordinate efferocytosis and internalize apoptotic cardiomyocytes remains unknown. The aim of this study was to determine whether SIRT3 (sirtuin-3) is required for both apoptotic cardiomyocyte engulfment and anti-inflammatory responses during efferocytosis. We generated myeloid SIRT3 knockout mice and FXN (frataxin) knock-in mice carrying an acetylation-defective lysine to arginine K189R mutation (FXNK189R). The mice were given Ang II (angiotensin II) infusion for 7 days. We analyzed cardiac macrophages' mitochondrial iron levels, efferocytosis activity, and phenotype both in vivo and in vitro. We showed that SIRT3 deficiency exacerbated Ang II-induced downregulation of the efferocytosis receptor MerTK (c-Mer tyrosine kinase) and proinflammatory cytokine production, accompanied by disrupted mitochondrial iron homeostasis in cardiac macrophages. Quantitative acetylome analysis revealed that SIRT3 deacetylated FXN at lysine 189. Ang II attenuated SIRT3 activity and enhanced the acetylation level of FXNK189. Acetylated FXN further reduced the synthesis of ISCs (iron-sulfur clusters), resulting in mitochondrial iron accumulation. Phagocytic internalization of apoptotic cardiomyocytes increased myoglobin content, and derived iron ions promoted mitochondrial iron overload and lipid peroxidation. An iron chelator deferoxamine improved the levels of MerTK and efferocytosis, thereby attenuating proinflammatory macrophage activation. FXNK189R mice showed improved macrophage efferocytosis, reduced cardiac inflammation, and suppressed cardiac fibrosis. The SIRT3-FXN axis has the potential to resolve cardiac inflammation by increasing macrophage efferocytosis and anti-inflammatory activities.

Inhibition of galectin-3 post-infarction impedes progressive fibrosis by regulating inflammatory profibrotic cascades.

Acute myocardial infarction (MI) causes inflammation, collagen deposition, and reparative fibrosis in response to myocyte death and, subsequently, a pathological myocardial remodelling process characterized by excessive interstitial fibrosis, driving heart failure (HF). Nonetheless, how or when to limit excessive fibrosis for therapeutic purposes remains uncertain. Galectin-3, a major mediator of organ fibrosis, promotes cardiac fibrosis and remodelling. We performed a preclinical assessment of a protein inhibitor of galectin-3 (its C-terminal domain, Gal-3C) to limit excessive fibrosis resulting from MI and prevent ventricular enlargement and HF. Gal-3C was produced by enzymatic cleavage of full-length galectin-3 or by direct expression of the truncated form in Escherichia coli. Gal-3C was intravenously administered for 7 days in acute MI models of young and aged rats, starting either pre-MI or 4 days post-MI. Echocardiography, haemodynamics, histology, and molecular and cellular analyses were performed to assess post-MI cardiac functionality and pathological fibrotic progression. Gal-3C profoundly benefitted left ventricular ejection fraction, end-systolic and end-diastolic volumes, haemodynamic parameters, infarct scar size, and interstitial fibrosis, with better therapeutic efficacy than losartan and spironolactone monotherapies over the 56-day study. Gal-3C therapy in post-MI aged rats substantially improved pump function and attenuated ventricular dilation, preventing progressive HF. Gal-3C in vitro treatment of M2-polarized macrophage-like cells reduced their M2-phenotypic expression of arginase-1 and interleukin-10. Gal-3C inhibited M2 polarization of cardiac macrophages during reparative response post-MI. Gal-3C impeded progressive fibrosis post-MI by down-regulating galectin-3-mediated profibrotic signalling cascades including a reduction in endogenous arginase-1 and inducible nitric oxide synthase (iNOS). Gal-3C treatment improved long-term cardiac function post-MI by reduction in the wound-healing response, and inhibition of inflammatory fibrogenic signalling to avert an augmentation of fibrosis in the periinfarct region. Thus, Gal-3C treatment prevented the infarcted heart from extensive fibrosis that accelerates the development of HF, providing a potential targeted therapy.

Circulating Gal-3 and sST2 are associated with acute exercise-induced sustained endothelial activation: Possible relevance for fibrosis development?

Long-term, intense endurance exercise training can occasionally induce endothelial micro-damage and cardiac fibrosis. The underlying mechanisms are incompletely understood. Twenty healthy, well-trained male participants (10runners and 10cyclists) performed a strenuous high-intensity interval training (HIIT) session matched by age, height, weight and maximal oxygen consumption. We assessed the acute exercise response of novel cardiac biomarkers of fibrosis [e.g., galectin-3 (Gal-3) and soluble suppression of tumorigenicity 2 (sST2)] per exercise modality and their relationship with haemodynamic contributors, such as preload, afterload and cardiac contractility index (CTi), in addition to endothelial damage by sustained activation and shedding of endothelial cells (ECs). Serum Gal-3 and sST2 concentrations were investigated by enzyme-linked immunosorbent assays; haemodynamics were analysed via impedance plethysmography and circulating ECs by flow cytometry. The Gal-3 and sST2 concentrations and ECs were elevated after exercise (P<0.001), without interaction between exercise modalities. Circulating Gal-3 and sST2 concentrations both showed a positive relationship with ECs (rrm =0.68, P=0.001 and rrm =0.57, P=0.010, respectively, both n=18). The EC association with Gal-3 was significant only in cyclists, but equally strong for both modalities. Gal-3 was also related to exercise-induced CTi (rrm =0.57, P=0.011, n=18). Cardiac wall stress is increased after an acute HIIT session but does not differ between exercise modalities. Exercise-released Gal-3 from cardiac macrophages could very probably drive systemic endothelial damage, based on an enhanced CTi. The importance of acute exercise-induced vascular resistances and cardiac contractility for the release of fibrotic biomarkers and any long-term pathological endothelial adaptation should be investigated further, also relative to the exercise modality. NEW FINDINGS: What is the central question of this study? Circulating biomarkers of cardiac wall stress and fibrosis are influenced by physical exercise. The underlying mechanisms per exercise modality are still unclear. What is the main finding and its importance? We show that galectin-3 (Gal-3) and soluble suppression of tumorigenicity 2 (sST2) are increased after acute exercise but do not differ between running and cycling. One haemodynamic contributor to the secretion of Gal-3 is an enhanced cardiac contractility. Acute exercise-released Gal-3 and sST2 are linked to sustained endothelial activation and cell shedding. This could be relevant in the context of fibrosis development and could identify athletes at risk for pathological endothelial adaptations.

Zanubrutinib Ameliorates Cardiac Fibrosis and Inflammation Induced by Chronic Sympathetic Activation.

(1) Background: Heart failure (HF) is the final stage of multiple cardiac diseases, which have now become a severe public health problem worldwide. β-Adrenergic receptor (β-AR) overactivation is a major pathological factor associated with multiple cardiac diseases and mediates cardiac fibrosis and inflammation. Previous research has demonstrated that Bruton's tyrosine kinase (BTK) mediated cardiac fibrosis by TGF-β related signal pathways, indicating that BTK was a potential drug target for cardiac fibrosis. Zanubrutinib, a second-generation BTK inhibitor, has shown anti-fibrosis effects in previous research. However, it is unclear whether Zanubrutinib can alleviate cardiac fibrosis induced by β-AR overactivation; (2) Methods: In vivo: Male C57BL/6J mice were treated with or without the β-AR agonist isoproterenol (ISO) to establish a cardiac fibrosis animal model; (3) Results: In vivo: Results showed that the BTK inhibitor Zanubrutinib (ZB) had a great effect on cardiac fibrosis and inflammation induced by β-AR. In vitro: Results showed that ZB alleviated β-AR-induced cardiac fibroblast activation and macrophage pro-inflammatory cytokine production. Further mechanism studies demonstrated that ZB inhibited β-AR-induced cardiac fibrosis and inflammation by the BTK, STAT3, NF-κB, and PI3K/Akt signal pathways both in vivo and in vitro; (4) Conclusions: our research provides evidence that ZB ameliorates β-AR-induced cardiac fibrosis and inflammation.

IL-30 protects against sepsis-induced myocardial dysfunction by inhibiting pro-inflammatory macrophage polarization and pyroptosis

Cardiac dysfunction is a well-recognized complication of sepsis and seriously affects the prognosis of sepsis patients. IL-30 has been reported to exert anti-inflammatory effects in various diseases. However, the role of IL-30 in sepsis-induced myocardial dysfunction (SIMD) remains unclear. Here, we explored the protective role of IL-30 in cecum ligation and puncture (CLP)-induced SIMD mice. IL-30 expression increased in the cardiac tissues of septic mice and was mainly derived from macrophages. IL-30 deletion or neutralization aggravated sepsis-induced cardiac dysfunction and injury, whereas recombinant IL-30 treatment significantly ameliorated it. Mechanistically, IL-30 deficiency exerts pro-inflammatory effects by promoting Ly6Chigh macrophage polarization and pyroptosis. Inhibiting NLRP3 with MCC950 significantly reversed cardiac dysfunction, macrophage polarization and pyroptosis aggravated by IL-30 deficiency. Recombinant IL-30 inhibited pro-inflammatory macrophage polarization and pyroptosis invivo and vitro. Taken together, these results suggest that IL-30 protects against SIMD by inhibiting pro-inflammatory macrophage polarization and pyroptosis.

Abstract P3074: Alkylating Chemotherapy Reshapes The Immune Landscape Of The Heart

Cancer survivors typically have concomitant cardiac comorbidities and were exposed to various chemotherapeutic agents. Despite extensive investigation, we still have a poor understanding of how chemotherapy agents impact cardiac physiology and disease pathogenesis. Alkylating agents, for instance, carboplatin, are the backbone of many chemotherapy regimens and have been applied in cancer treatment for decades. Cardiac macrophages are the primary component of the cardiac immune landscape and play essential roles in homeostasis and disease. My data implied that alkylating drugs induced cell death of the reparative cardiac macrophages (CCR2-) through activating apoptosis and necroptosis. However, the pro-inflammatory subset (CCR2+) is not decreased. Intriguingly, the reparative macrophages recovered four weeks after the carboplatin treatment through proliferation but showed different gene expression profiles. To investigate the functional influence of these reshaped macrophages in heart diseases, I established a hypertensive heart injury model and an ischemic heart injury model using carboplatin-exposed mice. Interestingly, these mice showed less fibrosis and smaller infarct size. Further, I used CD169-DTR mice that allow selective ablation of CCR2- macrophages. The attenuated remodeling phenotype is abolished without the reshaped CCR2- macrophages. Moreover, the type I interferon signaling in CCR2- macrophage is significantly upregulated in carboplatin-exposed mice upon hypertensive injury. Using neutralizing antibodies to block the IFN-I signaling, I found heart remodeling in carboplatin-treated mice became more advanced. Together, my study revealed that alkylating chemotherapy reshaped the immune landscape of the heart and mitigated adverse heart remodeling by activating the IFN-I signaling pathway in cardiac resident macrophages. These findings highlighted the role of CCR2- macrophages and the IFN-I signaling in the long-term cardiac effects of alkylating chemotherapy.

Abstract GS.102: Cd206 + Il-4rα + Macrophages Are Drivers Of Adverse Cardiac Remodeling In Ischemic Cardiomyopathy

Background: Cardiac macrophages are significantly expanded in chronic heart failure (HF); however, their role is unclear. We tested the hypothesis that CD206 + macrophages expressing interleukin (IL)-4Rα in the failing heart are key drivers of adverse left ventricular (LV) remodeling and ischemic cardiomyopathy. Methods and Results: Adult male C57BL/6 (WT) mice underwent non-reperfused myocardial infarction (MI) to induce HF, or sham operation, and cardiac macrophages were profiled using flow cytometry. As compared to sham hearts, both the frequency and absolute number of CD206 + macrophages steadily increased in post-MI hearts during the progression of LV remodeling and HF, such that at 8 w post-MI they comprised ~85% of macrophages. These macrophages were exclusively proliferative and predominantly C-C motif chemokine receptor (CCR2) - with nearly half expressing IL-4Rα, and correlated with LV dysfunction and fibrosis. IL-4-polarized bone marrow derived CD206 + macrophages exhibited marked upregulation of found in inflammatory zone (FIZZ)1 and induced FIZZ1-dependent myofibroblast differentiation of cardiac mesenchymal stem cells (cMSCs), in part related to DLL-4/Jagged1-Notch1 signaling. Intramyocardial adoptive transfer of M[IL-4], but not M[IL-10], macrophages induced progressive LV dilation and dysfunction and augmented cardiac fibrosis over 4 weeks in naïve mice. In vivo IL-4Rα gene silencing in mice with established HF effectively depleted cardiac CD206 + IL-4Rα + macrophages and induced reverse LV remodeling, improving fibrosis, neovascularization, and dysfunction, and suppressing local and systemic inflammation. Lastly, alternatively activated CD206 + and CD163 + macrophages were significantly expanded in human failing hearts and correlated with cardiac fibrosis, with the majority of CD163 + macrophages expressing IL-4Rα and FIZZ3, the human homolog of FIZZ1. Conclusion: CD206 + IL-4Rα + macrophages proliferate and expand in the failing heart and are key mediators of pathological remodeling and fibrosis, in part through the secretion of FIZZ1. Inhibition of CD206 + macrophage IL-4Rα signaling may be a fruitful therapeutic approach for alleviating LV remodeling in HF.

Abstract P3089: Selective Activation Of Cardiac Macrophages To Study Injury And Repair

Objective: Pathophysiological inflammatory responses play an important role during ischemia-mediated heart injury and remodeling, where monocytes and macrophages appear to be critically involved. Cardiac macrophages are generally classified as a recruited, monocyte-derived subset that is Ccr2 + and a tissue resident subset that is typically Ccr2 - . Recent studies have shown that these two subsets have divergent functions with Ccr2 - macrophages being pro-regenerative while Ccr2 + macrophages are pro-inflammatory. Here we selectively manipulated the content and/or activation of these two macrophage subsets in the heart. Methods and Results: To study the function of Ccr2 + cardiac macrophages in the heart, we have overexpressed Ccl-2 (Mcp-1) ligand specifically in cardiomyocytes by using adeno-associated virus serotype-9 (AAV9) mediated gene transfer. Postnatal day 4 (p4) Ccr2 RFP/+ pups were injected with either AAV9-Ccl-2 or AAV9-control. At two months of age, we observed increased Ccl-2 transcripts and protein expression in the heart as measured by qRT-PCR and ELISA, respectively. By quantifying immune cells using flow cytometry, we observed increased infiltration of Ccr2 + macrophages (CD45 + CD11b + CD64 + ) in the hearts of AAV9-Ccl2 injected mice compared to controls. However, echocardiography analysis revealed no change in cardiac function in adult Ccl-2 overexpressed mice. Moreover, the recruitment of Ccr2 + macrophages into the myocardium did not cause fibroblast expansion or activation. Conclusion: Our results show that cardiomyocyte-specific overexpression of Ccl-2 induces profound infiltration of Ccr2 + macrophages into the myocardium but this did not alter cardiac function or cause fibrosis at two or six months of age.

Abstract P3087: Macrophage Fatty Acid Metabolism Promotes Splenic Hematopoiesis Through Mitochondrial Signaling During Heart Failure With Preserved Ejection Fraction

Rationale: Diastolic dysfunction, including during heart failure with preserved ejection fraction (HFpEF), is a widespread morbidity with limited therapeutic options. Though heterogeneous in etiology, major risk factors include hyperlipidemia, hypertension, and inflammation. Recent studies have also shown significant changes in systemic metabolite availability during HFpEF, further suggesting that metabolic changes may be an important indicator of HFpEF pathophysiology. Macrophages, which have previously been implicated in the promotion of diastolic dysfunction, utilize specific metabolic pathways to drive inflammation or tissue repair. However, little is understood about myeloid metabolism during HFpEF. Objective: We set out to define the functional and metabolic heterogeneity of the cardiac immune response during diastolic dysfunction. Methods/Results: Diastolic dysfunction was induced by HFD and eNOS inhibition in mice. scRNAseq showed a specific induction of pro-fibrotic and lipid handling pathways in cardiac macrophages, including the fatty acid receptor Cd36. In live animals, myeloid deficiency of Cd36 prevented diastolic dysfunction and was associated with dampened cardiac inflammation. Investigating immune cell sourcing during HFpEF revealed that a splenic hematopoietic niche formed in wild-type mice. Further, mitochondrial metabolism in splenic macrophages was associated with increased expression of VCAM-1, the latter of which has been shown to play a role in retention of HSCs in the spleen. Indeed, blocking VCAM-1 or genetic deletion of VCAM-1 in myeloid cells was sufficient to disrupt retention of HSCs within the spleen during HFpEF. Culturing splenic macrophages with saturated fatty acids (SFAs) in vitro was sufficient to induce expression of Vcam1. Increased cellular citrate levels were correlated with increased histone acetylation at the Vcam1 promoter in splenic macrophages cultured with SFAs. Inhibition of mitochondrial uptake of fatty acids through CPT1a siRNA or inhibition of ACLY were sufficient to prevent this upregulation of Vcam1 expression. Conclusions: Our data suggest a critical role for myeloid metabolism in the sourcing of detrimental-monocyte-derived macrophages from the spleen to the heart.

Abstract P3085: The Macrophage Landscape Across The Lifespan Of A Human Cardiac Allograft

Much of our knowledge of organ rejection after transplantation is derived from rodent studies. We used single-nucleus RNA sequencing (snRNA-seq) to investigate the inflammatory milieu in pediatric cardiac allografts at different stages post transplantation. We distinguished donor from recipient-derived cardiac cells using naturally occurring genetic variants embedded in snRNA-seq data. Donor-derived cardiac resident macrophages dMPs, which accompany the cardiac allograft into the recipient, are rapidly lost post transplantation. On the other hand, monocyte-derived macrophages from the recipient (rMPs) quickly populate the heart post transplantation and form two macrophage populations, rMP1 and rMP2. While rMP2s have cell signatures similar to dMPs, they lack pro-reparative phagocytic activity and instead express profibrotic genes. In contrast, rMP1 express genes consistent with hallmarks of cellular rejection. Our data further support the conclusion that rMP1s activate a subset of natural killer (NK) cells, turning them into a cytotoxic cell population. Self-amplifying reciprocal signaling occurs between rMP1s and NKs, which is consistent with a key mechanism for clinical rejection. Our data reveal an imbalance of donor-derived and recipient-derived macrophages in the pediatric cardiac allograft after heart transplantation. Moreover, we observed a clear functional dichotomy of recipient-derived macrophages. At different stages after heart transplantation, rMPs contribute to acute adverse damage to the myocardium and chronic organ failure.

Abstract P1127: Induced Pluripotent Stem Cell-derived Macrophages Modulate Human Engineered Cardiac Tissue Function

In the native human myocardium, cardiac resident macrophages are a critical mediator for normal cardiac function, performing a variety of biological roles in electrical conduction, regeneration, and phagocytosis and efferocytosis. However, most models of human engineered cardiac tissues (hECT), which aim to recapitulate the native adult myocardium for disease modeling and drug screening applications, do not include a macrophage population. Thus, in this study we introduced an iPSC-derived macrophage (iM0) population into an all iPSC-derived tissue engineered model of the human myocardium in order to assess its impact on tissue phenotype and function. CD14 + macrophages were differentiated from iPSCs and added as 7% of the total cell composition (7% iM0, 69.75% iCM, 23.25% iCF) in the hECT model. The hECTs were then cultured for 4 weeks and subjected to a ramped electrical stimulation protocol for matured function. The resulting tissues showed that the presence of a CD68 + macrophage population was maintained over this extended period of culture and significantly impacted hECT function, increasing contractile force production and modulating contraction and relaxation velocity. Interrogation of calcium signaling via isoproterenol revealed that iM0 presence in the tissues may be causing these functional differences via modulation of the cAMP signaling pathway. These observations demonstrate that macrophages significantly contribute to cardiac function in iPSC-derived hECT models and emphasize the need to further explore their contributions not only in healthy hECT models, but also in the contexts of disease and injury.

Abstract P3098: Immune Suppression Ameliorates A Mouse Model Of Lmna-related Cardiomyopathy With Nuclear Envelope Rupture

Lamin A/C (encoded by the LMNA gene) are the components of the nuclear lamina, a protein meshwork providing the structural integrity to the nuclear membrane. Dilated cardiomyopathy (DCM) caused by LNNA gene mutations is the second most common familial DCM in adults and associated with severe prognosis and high mortality. Despite decades of investigations into cell autonomous impacts of LMNA mutations, LMNA-DCM lacks effective treatments except for heart transplantation. Here, we identify cell non-autonomous effects of LMNA mutations and demonstrate the efficacy of targeting immune activities in mouse models of LMNA-DCM. We induced Lmna gene deletion specifically in cardiomyocytes in adult mice and tracked molecular, cellular, and physiological alterations in the heart at a high temporal resolution. We identified frequent localized nuclear envelope rupture, with the concomitant exposure of nuclear DNA to the cytoplasm, as the earliest direct consequence of Lamin A/C protein reduction in cardiomyocytes. Surprisingly, nuclear envelope rupture did accompany cardiomyocyte cell death or extensive transcriptional changes or cytosolic DNA sensing pathway activation within the cardiomyocytes. Instead, it coincided with extensive proinflammatory signaling activation within the cardiac macrophage population. This was followed by extensive fibrosis and structural and functional alterations of the heart, culminating in DCM and death. Treatment of LMNA-DCM mice with the immunosuppressant Dexamethasone suppressed fibrosis, profoundly improved the cardiac structure and function, and conferred a two-fold lifespan extension after Lmna gene deletion. This work suggested that immune activation is the major pathological component of LMNA-DCM and an effective target for treating LMNA-DCM patients.

Abstract P1065: Primitive Macrophages Enhance The Function Of Bioengineered Human Cardiac Microtissues

Human cardiac bioengineering has traditionally focused on cardiomyocyte differentiation and maturation. Unlike normal cardiac growth in the native human heart, human pluripotent stem cell (hPSC)- derived cardiomyocytes are typically grown in the absence of key supportive populations, which may negatively impact function due to a failure to recreate complex multicellular environments. In vivo murine studies have demonstrated that embryonic-derived resident cardiac macrophages promote neonatal cardiac regeneration, yet macrophages have not been incorporated in cardiac bioengineering approaches. We hypothesized that inclusion of human embryonic stem cell derived primitive macrophages (hESC-macrophages) into bioengineered human cardiac microtissues (“Biowires”) would improve cardiomyocyte functional properties. We found that addition of hESC-macrophages into Biowires led to enhanced contractile force, improved relaxation, reduced excitation threshold, increased maximum capture rate, and increased conduction velocity that mechanistically was correlated with improved calcium cycling, indicating hESC-macrophages improve a broad range of electromechanical properties. Moreover, hESC-macrophages enhanced microtissue function across cardiomyocyte cell sources and bioengineering platforms (Experimental N=6 independent cellular differentiations across 2 different platforms). These changes occurred in the absence of significant transcriptional shifts in cardiomyocytes. Rather, Biowires with hESC-macrophages had reduced mitochondrial DNA release, oxidative stress and cytotoxicity in the very early phases of tissue formation, thereby promoting a favorable environment for cardiac microtissue development. In conclusion, these data reveal a previously unappreciated, yet major beneficial role for macrophages specifically in human cardiac tissue engineering. In general, bioengineering human tissue continues to be a major research focus, and our data argue that inclusion of yolk sac-derived macrophages may improve early microtissue formation and modeling in vitro , and could enhance cell delivery approaches in vivo .

Cardiac macrophages undergo dynamic changes after coxsackievirus B3 infection and promote the progression of myocarditis.

Although most patients with acute viral myocarditis recover spontaneously, some patients progress to heart failure. Perturbations in innate immunity may partially explain the heterogeneity of clinical outcomes. As the most abundant immune cells in the heart, cardiac macrophages have heterogeneous origins, including embryonic-derived resident macrophages (ResMϕs) and monocyte-derived macrophages (MoMFs). However, the time course change and role of cardiac macrophage subsets has not been fully explored. In the present study, we found that BALB/c mice had prolonged MoMF accumulation and low proportions of ResMϕs that could not be restored to normal levels. MoMFs of BALB/c mice generally exhibit an M1-dominant functional phenotype. Moreover, the preferential depletion of MoMF by a C-C chemokine receptor type 2 (CCR2) inhibitor resulted in improved acute myocarditis and chronic fibrosis, as well as the recovery of ResMϕs number and reduced CD4+ T cell expansion. Hence, immunomodulatory therapy that targets the balance among cardiac macrophages and modulates their function is expected to prevent the progression of cardiac injury to overt heart failure and improve adverse outcomes.

Recruited and resident cardiac macrophage phenotypes converge in the healing infarct but not in the failing heart exposed to continuous pressure overload

Recruited and resident cardiac macrophage phenotypes converge in the healing infarct but not in the failing heart exposed to continuous pressure overload

Comparative single-cell profiling reveals distinct cardiac resident macrophages essential for zebrafish heart regeneration.

Zebrafish exhibit a robust ability to regenerate their hearts following injury, and the immune system plays a key role in this process. We previously showed that delaying macrophage recruitment by clodronate liposome (-1d_CL, macrophage-delayed model) impairs neutrophil resolution and heart regeneration, even when the infiltrating macrophage number was restored within the first week post injury (Lai et al., 2017). It is thus intriguing to learn the regenerative macrophage property by comparing these late macrophages vs. control macrophages during cardiac repair. Here, we further investigate the mechanistic insights of heart regeneration by comparing the non-regenerative macrophage-delayed model with regenerative controls. Temporal RNAseq analyses revealed that -1d_CL treatment led to disrupted inflammatory resolution, reactive oxygen species homeostasis, and energy metabolism during cardiac repair. Comparative single-cell RNAseq profiling of inflammatory cells from regenerative vs. non-regenerative hearts further identified heterogeneous macrophages and neutrophils, showing alternative activation and cellular crosstalk leading to neutrophil retention and chronic inflammation. Among macrophages, two residential subpopulations (hbaa+ Mac and timp4.3+ Mac 3) were enriched only in regenerative hearts and barely recovered after +1d_CL treatment. To deplete the resident macrophage without delaying the circulating macrophage recruitment, we established the resident macrophage-deficient model by administrating CL earlier at 8 d (-8d_CL) before cryoinjury. Strikingly, resident macrophage-deficient zebrafish still exhibited defects in revascularization, cardiomyocyte survival, debris clearance, and extracellular matrix remodeling/scar resolution without functional compensation from the circulating/monocyte-derived macrophages. Our results characterized the diverse function and interaction between inflammatory cells and identified unique resident macrophages prerequisite for zebrafish heart regeneration.

Ezh2 emerges as an epigenetic checkpoint regulator during monocyte differentiation limiting cardiac dysfunction post-MI

Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative immunoregulatory M2-like macrophage polarization; known to impact cardiac repair after Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time an ectopic EZH2, and putative, cytoplasmic inactive localization of the epigenetic enzyme, during monocyte differentiation into M2 macrophages in vitro as well as in immunomodulatory cardiac macrophages in vivo in the post-MI acute inflammatory phase. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation of bivalent gene promoters, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction in female mice post-MI in vivo. In conclusion, our study reveals that pharmacological epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.