Distinct roles of resident and nonresident macrophages in nonischemic cardiomyopathy.

Nonischemic cardiomyopathy (NICM) resulting from long-standing hypertension, valvular disease, and genetic mutations is a major cause of heart failure worldwide. Recent observations suggest that myeloid cells can impact cardiac function, but the role of tissue-intrinsic vs. tissue-extrinsic myeloid cells in NICM remains poorly understood. Here, we show that cardiac resident macrophage proliferation occurs within the first week following pressure overload hypertrophy (POH; a model of heart failure) and is requisite for the heart's adaptive response. Mechanistically, we identify Kruppel-like factor 4 (KLF4) as a key transcription factor that regulates cardiac resident macrophage proliferation and angiogenic activities. Finally, we show that blood-borne macrophages recruited in late-phase POH are detrimental, and that blockade of their infiltration improves myocardial angiogenesis and preserves cardiac function. These observations demonstrate previously unappreciated temporal and spatial roles for resident and nonresident macrophages in the development of heart failure.

Regional variations in ex-vivo diffusion tensor anisotropy are associated with cardiomyocyte remodeling in rats after left ventricular pressure overload.

BACKGROUND: Pressure overload left ventricular (LV) hypertrophy is characterized by increased cardiomyocyte width and ventricle wall thickness, however the regional variation of this remodeling is unclear. Cardiovascular magnetic resonance (CMR) diffusion tensor imaging (DTI) may provide a non-invasive, comprehensive, and geometrically accurate method to detect regional differences in structural remodeling in hypertrophy. We hypothesized that DTI parameters, such as fractional and planar anisotropy, would reflect myocyte remodeling due to pressure overload in a regionally-dependent manner. METHODS: We investigated the regional distributions of myocyte remodeling in rats with or without transverse aortic constriction (TAC) via direct measurement of myocyte dimensions with confocal imaging of thick tissue sections, and correlated myocyte cross-sectional area and other geometric features with parameters of diffusivity from ex-vivo DTI in the same regions of the same hearts. RESULTS: We observed regional differences in several parameters from DTI between TAC hearts and SHAM controls. Consistent with previous studies, helix angles from DTI correlated strongly with those measured directly from histological sections (p < 0.001, R2 = 0.71). There was a transmural gradient in myocyte cross-sectional area in SHAM hearts that was diminished in the TAC group. We also found several regions of significantly altered DTI parameters in TAC LV compared to SHAM, especially in myocyte sheet angle dispersion and planar anisotropy. Among others, these parameters correlated significantly with directly measured myocyte aspect ratios. CONCLUSIONS: These results show that structural remodeling in pressure overload LV hypertrophy is regionally heterogeneous, especially transmurally, with a greater degree of remodeling in the sub-endocardium compared to the sub-epicardium. Additionally, several parameters derived from DTI correlated significantly with measurements of myocyte geometry from direct measurement in histological sections. We suggest that DTI may provide a non-invasive, comprehensive method to detect regional structural myocyte LV remodeling during disease.

Cardiac-restricted overexpression or deletion of tissue inhibitor of matrix metalloproteinase-4: differential effects on left ventricular structure and function following pressure overload-induced hypertrophy.

Historically, the tissue inhibitors of matrix metalloproteinases (TIMPs) were considered monochromatic in function. However, differential TIMP profiles more recently observed with left ventricular (LV) dysfunction and matrix remodeling suggest more diverse biological roles for individual TIMPs. This study tested the hypothesis that cardiac-specific overexpression (TIMP-4OE) or deletion (knockout; TIMP-4KO) would differentially affect LV function and structure following pressure overload (LVPO). LVPO (transverse aortic constriction) was induced in mice (3.5 ± 0.1 mo of age, equal sex distribution) with TIMP-4OE (n = 38), TIMP-4KO (n = 24), as well as age/strain-matched wild type (WT, n = 25), whereby indexes of LV remodeling and function such as LV mass and ejection fraction (LVEF) were determined at 28 days following LVPO. Following LVPO, both early (7 days) and late (28 days) survival was ~25% lower in the TIMP-4KO group (P < 0.05). While LVPO increased LV mass in all groups, the relative hypertrophic response was attenuated with TIMP-4OE. With LVPO, LVEF was similar between WT and TIMP-4KO (48 ± 2% and 45 ± 3%, respectively) but was higher with TIMP-4OE (57 ± 2%, P < 0.05). With LVPO, LV myocardial collagen expression (type I, III) increased by threefold in all groups (P < 0.05), but surprisingly this response was most robust in the TIMP-4KO group. These unique findings suggest that increased myocardial TIMP-4 in the context of a LVPO stimulus may actually provide protective effects with respect to survival, LV function, and extracellular matrix (ECM) remodeling. These findings challenge the canonical belief that increased levels of specific myocardial TIMPs, such as TIMP-4 in and of themselves, contribute to adverse ECM accumulation following a pathological stimulus, such as LVPO.

Cardiac sympathetic overdrive, M2 macrophage activation and fibroblast heterogeneity are associated with cardiac remodeling in a chronic pressure overload rat model of HFpEF.

Heart failure with preserved ejection fraction (HFpEF) is a multifaceted pathogenesis disease and the exact mechanisms driving HFpEF have not been completely elucidated. Pressure overload hypertrophy (POH) related fibroblasts and M2 macrophages in HFpEF myocardium have been recently identified and are now of great interest. Sympathetic overdrive has also been implicated in HFpEF. This study is designed to dynamically observe the potential roles of aforementioned mechanisms in pathological remodeling and cardiac dysfunction in chronic PO rats. Surgical constriction of the abdominal aorta was used for induction of HFpEF. Echocardiography, electrocardiogram, hemodynamic measurement, hematoxylin and eosin staining, Masson staining, immunohistochemistry and immunofluorescence were performed to assess the changes in heart dysfunction, cardiac remodeling and driving mechanisms at different time points (2, 18, 24 weeks). The PO induced HFpEF model was well established, which was confirmed by the persistent increase in carotid artery systolic and diastolic blood pressure, and left ventricle hypertrophy at the corresponding postoperative stage. Meanwhile, PO hypertrophy gradually developed into HFpEF, associated with QT and QTc intervals prolongation, normal systolic (EF was maintained at >50%) but impaired diastolic function (increasing LVEDP and LV -dP/dtmin, abnormal E/A ratio), increased myocytes size, and observed relatively slight inflammatory infiltration but robust reactive fibrosis. IHC staining further confirmed that macrophages (CD68) but not neutrophils (MPO) or T cells (CD3) accounted for a predominant proportion of infiltrating cells. Mechanistically, we found that the infiltrating macrophages in the heart expressed high levels of CD206 which was simultaneously adjacent to POH fibroblasts appeared to overexpression of α-SMA in PO rats at late stages. Interestingly, we distinguished two different POHF sub-populations during PO induced HFpEF development, according to non overlapping signals of α-SMA and PDGFRα/ß proteins. Additionally, PO led to a pronounced exaggeration in sympathetic fibers at all time points. These findings suggest that the establishing model here begins with cardiac sympathetic overdrive, subsequently along with immune cells especially M2 macrophage accumulation and fibroblast heterogeneity at later stages is associated with the development of cardiac maladaptive remodeling and diastolic dysfunction thus further progression to HFpEF.

Chronic pressure-overload hypertrophy attenuates vortex formation time in patients with severe aortic stenosis and preserved left ventricular systolic function undergoing aortic valve replacement.

OBJECTIVE: Transmitral blood flow produces a vortex ring that enhances the hydraulic efficiency of early left ventricular (LV) filling. The effect of pressure-overload hypertrophy on the duration of LV vortex ring formation (vortex formation time [VFT]) is unknown. The current investigation tested the hypothesis that chronic LV pressure-overload hypertrophy produced by severe aortic stenosis (AS) reduces VFT in patients with preserved LV systolic function undergoing aortic valve replacement. DESIGN: Observational study. SETTING: Veterans Affairs Medical Center. PARTICIPANTS: After the Institutional Review Board's approval, 8 patients (7 men and 1 woman; age, 62±5 y; and ejection fraction, 59%±5%) with AS (peak pressure gradient, 81±22 mmHg; aortic valve area, 0.78±0.25 cm(2)) scheduled for aortic valve replacement were compared with 8 patients (all men; age, 63±3 y; and ejection fraction, 60%±7%) without AS undergoing coronary artery bypass graft surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Under general anesthesia, peak early LV filling (E) and atrial systole (A) blood flow velocities and their corresponding velocity-time integrals were obtained using pulse-wave Doppler echocardiography to determine E/A and atrial filling fraction (ß). Mitral valve diameter (D) was calculated as the average of minor and major axis lengths obtained in the midesophageal bicommissural and long-axis transesophageal echocardiography imaging planes, respectively. Posterior wall thickness (PWT) was measured at end-diastole using M-mode echocardiography. VFT was calculated as 4×(1-ß)×SV/πD(3), where SV = stroke volume measured using thermodilution. Systemic and pulmonary hemodynamics, LV diastolic function, PWT, and VFT were determined during steady-state conditions 30 minutes before cardiopulmonary bypass. Early LV filling was attenuated in patients with AS (eg, E/A, 0.77±0.11 compared with 1.23±0.13; ß, 0.43±0.09 compared with 0.35±0.02; p<0.05 for each). LV hypertrophy was observed (PWT, 1.4±0.1 cm compared with 1.1±0.2 cm; p<0.05) and VFT was lower (3.0±0.9 v 4.3±0.5; p<0.05) in patients with versus without AS. Linear regression analysis showed a significant correlation between VFT and PWT (VFT = -2.57 ×PWT + 6.81; r(2) = 0.345; p = 0.017). CONCLUSION: The results indicated that pressure-overload hypertrophy produced by AS reduced VFT in patients with normal LV systolic function undergoing aortic valve replacement.

The GABAA Receptor Influences Pressure Overload-Induced Heart Failure by Modulating Macrophages in Mice.

Background: Myocardial macrophages have key roles in cardiac remodeling and dysfunction. The gamma-aminobutyric acid subtype A (GABAA) receptor was recently found to be distributed in macrophages, allowing regulation of inflammatory responses to various diseases. This study aimed to clarify the role of GABAA receptor-mediated macrophage responses in pressure overload-induced heart failure. Methods and Results: C57BL/6J mice underwent transverse aortic constriction for pressure-overload hypertrophy (POH) and were intraperitoneally treated with a specific GABAA receptor agonist (topiramate) or antagonist (bicuculline). Echocardiography, histology, and flow cytometry were performed to evaluate the causes and effects of myocardial hypertrophy and fibrosis. Activation of the GABAA receptor by topiramate reduced ejection fraction and fractional shortening, enlarged the end-diastolic and end-systolic left ventricular internal diameter, aggravated myocardial hypertrophy and fibrosis, and accelerated heart failure in response to pressure overload. Mechanistically, topiramate increased the number of Ly6Clow macrophages in the heart during POH and circulating Ly6Chigh classic monocyte infiltration in late-phase POH. Further, topiramate drove Ly6Clow macrophages toward MHCIIhigh macrophage polarization. As a result, Ly6Clow macrophages activated the amphiregulin-induced AKT/mTOR signaling pathway, and Ly6ClowMHCIIhigh macrophage polarization increased expression levels of osteopontin and TGF-ß, which led to myocardial hypertrophy and fibrosis. Conversely, GABAA receptor blockage with bicuculline reversed these effects. Conclusions: Control of the GABAA receptor activity in monocytes/macrophages plays an important role in myocardial hypertrophy and fibrosis after POH. Blockade of the GABAA receptor has the potential to improve pressure overload-induced heart failure.

Corrigendum: The GABAA Receptor Influences Pressure Overload-Induced Heart Failure by Modulating Macrophages in Mice.

[This corrects the article DOI: 10.3389/fimmu.2021.670153.].

Temporal regulation of protein O-GlcNAc levels during pressure-overload cardiac hypertrophy.

Protein posttranslational modifications (PTMs) by O-linked ß-N-acetylglucosamine (O-GlcNAc) rise during pressure-overload hypertrophy (POH) to affect hypertrophic growth. The hexosamine biosynthesis pathway (HBP) branches from glycolysis to make the moiety for O-GlcNAcylation. It is speculated that greater glucose utilization during POH augments HBP flux to increase O-GlcNAc levels; however, recent results suggest glucose availability does not primarily regulate cardiac O-GlcNAc levels. We hypothesize that induction of key enzymes augment protein O-GlcNAc levels primarily during active myocardial hypertrophic growth and remodeling with early pressure overload. We further speculate that downregulation of protein O-GlcNAcylation inhibits ongoing hypertrophic growth during prolonged pressure overload with established hypertrophy. We used transverse aortic constriction (TAC) to create POH in C57/Bl6 mice. Experimental groups were sham, 1-week TAC (1wTAC) for early hypertrophy, or 6-week TAC (6wTAC) for established hypertrophy. We used western blots to determine O-GlcNAc regulation. To assess the effect of increased protein O-GlcNAcylation with established hypertrophy, mice received thiamet-g (TG) starting 4 weeks after TAC. Protein O-GlcNAc levels were significantly elevated in 1wTAC versus Sham with a fall in 6wTAC. OGA, which removes O-GlcNAc from proteins, fell in 1wTAC versus sham. GFAT is the rate-limiting HBP enzyme and the isoform GFAT1 substantially rose in 1wTAC. With established hypertrophy, TG increased protein O-GlcNAc levels but did not affect cardiac mass. In summary, protein O-GlcNAc levels vary during POH with elevations occurring during active hypertrophic growth early after TAC. O-GlcNAc levels appear to be regulated by changes in key enzyme levels. Increasing O-GlcNAc levels during established hypertrophy did not restart hypertrophic growth.

Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation.

Heart failure remains a major cause of hospitalization and death worldwide. Heart failure can be caused by abnormalities in the epigenome resulting from dysregulation of histone-modifying enzymes. While chromatin enzymes catalyzing lysine acetylation and methylation of histones have been the topic of many investigations, the role of arginine methyltransferases has been overlooked. In an effort to understand regulatory mechanisms implicated in cardiac hypertrophy and heart failure, we assessed the expression of protein arginine methyltransferases (PRMTs) in the left ventricle of failing human hearts and control hearts. Our results show a significant up-regulation of protein arginine methyltransferase 6 (PRMT6) in failing human hearts compared to control hearts, which also occurs in the early phase of cardiac hypertrophy in mouse hearts subjected to pressure overload hypertrophy induced by trans-aortic constriction (TAC), and in neonatal rat ventricular myocytes (NRVM) stimulated with the hypertrophic agonist phenylephrine (PE). These changes are associated with a significant increase in arginine 2 asymmetric methylation of histone H3 (H3R2Me2a) and reduced lysine 4 tri-methylation of H3 (H3K4Me3) observed both in NRVM and in vivo. Importantly, forced expression of PRMT6 in NRVM enhances the expression of the hypertrophic marker, atrial natriuretic peptide (ANP). Conversely, specific silencing of PRMT6 reduces ANP protein expression and cell size, indicating that PRMT6 is critical for the PE-mediated hypertrophic response. Silencing of PRMT6 reduces H3R2Me2a, a mark normally associated with transcriptional repression. Furthermore, evaluation of cardiac contractility and global ion channel activity in live NRVM shows a striking reduction of spontaneous beating rates and prolongation of extra-cellular field potentials in cells expressing low-level PRMT6. Altogether, our results indicate that PRMT6 is a critical regulator of cardiac hypertrophy, implicating H3R2Me2a as an important histone modification. This study identifies PRMT6 as a new epigenetic regulator and suggests a new point of control in chromatin to inhibit pathological cardiac remodeling.

Tet2-Mediated Clonal Hematopoiesis Accelerates Heart Failure Through a Mechanism Involving the IL-1ß/NLRP3 Inflammasome.

BACKGROUND: Recent studies have shown that hematopoietic stem cells can undergo clonal expansion secondary to somatic mutations in leukemia-related genes, thus leading to an age-dependent accumulation of mutant leukocytes in the blood. This somatic mutation-related clonal hematopoiesis is common in healthy older individuals, but it has been associated with an increased incidence of future cardiovascular disease. The epigenetic regulator TET2 is frequently mutated in blood cells of individuals exhibiting clonal hematopoiesis. OBJECTIVES: This study investigated whether Tet2 mutations within hematopoietic cells can contribute to heart failure in 2 models of cardiac injury. METHODS: Heart failure was induced in mice by pressure overload, achieved by transverse aortic constriction or chronic ischemia induced by the permanent ligation of the left anterior descending artery. Competitive bone marrow transplantation strategies with Tet2-deficient cells were used to mimic TET2 mutation-driven clonal hematopoiesis. Alternatively, Tet2 was specifically ablated in myeloid cells using Cre recombinase expressed from the LysM promoter. RESULTS: In both experimental heart failure models, hematopoietic or myeloid Tet2 deficiency worsened cardiac remodeling and function, in parallel with increased interleukin-1beta (IL-1ß) expression. Treatment with a selective NLRP3 inflammasome inhibitor protected against the development of heart failure and eliminated the differences in cardiac parameters between Tet2-deficient and wild-type mice. CONCLUSIONS: Tet2 deficiency in hematopoietic cells is associated with greater cardiac dysfunction in murine models of heart failure as a result of elevated IL-1ß signaling. These data suggest that individuals with TET2-mediated clonal hematopoiesis may be at greater risk of developing heart failure and respond better to IL-1ß-NLRP3 inflammasome inhibition.

Transmural gradients of myocardial structure and mechanics: Implications for fiber stress and strain in pressure overload.

Although a truly complete understanding of whole heart activation, contraction, and deformation is well beyond our current reach, a significant amount of effort has been devoted to discovering and understanding the mechanisms by which myocardial structure determines cardiac function to better treat patients with cardiac disease. Several experimental studies have shown that transmural fiber strain is relatively uniform in both diastole and systole, in contrast to predictions from traditional mechanical theory. Similarly, mathematical models have largely predicted uniform fiber stress across the wall. The development of this uniform pattern of fiber stress and strain during filling and ejection is due to heterogeneous transmural distributions of several myocardial structures. This review summarizes these transmural gradients, their contributions to fiber mechanics, and the potential functional effects of their remodeling during pressure overload hypertrophy.

Papel relativo da remodelação geométrica do ventrículo esquerdo, morfológica e funcional do miocárdio na transição da hipertrofia compensada para a falência cardíaca em ratos com estenose aórtica supravalvar / Relative role of left ventricular geometric remodeling and of morphological and functional myocardial remodeling in the transition from compensated hypertrophy to heart failure in rats with supravalvar aortic stenosis

OBJETIVO: Avaliar a contribuição relativa da remodelação geométrica do ventrículo esquerdo (VE) e das alterações morfológicas e funcionais do miocárdio, em ratos com estenose aórtica supravalvar (EAS), na fase de transição da hipertrofia compensada para a insuficiência cardíaca congestiva (ICC). MÉTODOS: Vinte e uma semanas após a indução da EAS os ratos foram classificados como controles (GC,n=13), não portadores (GE,n=11) ou portadores de insuficiência cardíaca congestiva (GE-IC,n=12).Todos os grupos foram avaliados com estudo ecocardiográfico, hemodinâmico e morfológico do miocárdio. RESULTADOS: Vinte e uma semanas após EAS: índice de massa (GE-IC>GE>GC,p<0.05); pressão sistólica: (GE-IC = GE>GC, p<0,05); pressão diastólica: (GE-IC>GE>GC, p<0,05); estresse meridional sistólico e diastólico: (GE-IC>GE>GC,p<0.05); área de secção dos miócitos: (GE-IC>GE>GC, p<0,05) e conteúdo de hidroxiprolina: (GE-IC>GE>GC, p<0,05) do VE. No grupo GE-IC o remodelamento geométrico do VE foi caracterizado por aumento significante das dimensões e espessura relativa da parede normal (remodelamento excêntrico) enquanto que o grupo GE apresentou remodelamento concêntrico. Os índices de desempenho do VE do grupo GE-IC foram significantemente menores que do grupo GE. CONCLUSÃO: Os grupos GE-IC e GE diferiram primariamente no processo de remodelação geométrica do VE e estrutural do miocárdio que estabeleceu um estado cronicamente compensado no grupo GE e precipitou a ICC no grupo GE-IC na vigência de graus equivalentes de comprometimento da contratilidade. Neste modelo experimental a fase de transição da hipertrofia compensada para a ICC está mais estreitamente relacionada com o remodelamento geométrico adverso do VE e estrutural do miocárdio do que com o grau de comprometimento da contratilidade.

OBJECTIVE: To evaluate the relative contribution of left ventricular (LV) geometric remodeling and of morphological and functional myocardial changes in rats with induced supravalvar aortic stenosis (SAS), in the transition from compensated hypertrophy to congestive heart failure (CHF). METHODS: Twenty one weeks after induction of SAS, the rats were classified as controls (CG, n=13), without congestive heart failure (SG, n=11), or with congestive heart failure (SG-HF, n=12). All groups were evaluated with echocardiographic, hemodynamic and morphological study of the myocardium. RESULTS: Twenty one weeks after SAS: mass index (SG-HF>SG>CG, p<0.05); systolic pressure (SG-HF= SG>CG, p<0.05); diastolic pressure (SG-HF>SG>CG, p<0.05); systolic and diastolic meridional stress (SG-HF>SG>CG, p<0.05); LV myocyte cross-sectional area (SG-HF>SG>CG, p<0.05) and hydroxyproline content (SG-HF>SG>CG, p<0.05). In the SG-HF group, LV geometric remodeling was characterized by a significant increase in dimensions and relative thickness of the normal wall (excentric remodeling), whereas the SG group presented a concentric remodeling. Indexes of LV performance in the SG-HF group were significantly lower than those of the SG group. CONCLUSION: The SG-HF and SG groups differed primarily in the LV geometric remodeling and structural myocardial remodeling process, which established a chronically compensated state in the SG group and triggered CHF in the SG-HF group in the presence of equivalent degrees of impaired contractility.