Characterization of anthracycline-induced cardiotoxicity by diffusion tensor magnetic resonance imaging.

Anthracyclines are highly potent anti-cancer drugs, but their clinical use is limited by severe cardiotoxic side effects. The impact of anthracycline-induced cardiotoxicity (AIC) on left ventricular (LV) microarchitecture and diffusion properties remains unknown. This study sought to characterize AIC by cardiovascular magnetic resonance diffusion tensor imaging (DTI). Mice were treated with Doxorubicin (DOX; n = 16) for induction of AIC or saline as corresponding control (n = 15). Cardiac function was assessed via echocardiography at the end of the study period. Whole hearts (n = 8 per group) were scanned ex vivo by high-resolution DTI at 7 T. Results were correlated with histopathology and mass spectrometry imaging. Mice with AIC demonstrated systolic dysfunction (LVEF 52 ± 3% vs. 43 ± 6%, P < 0.001), impaired global longitudinal strain (-19.6 ± 2.0% vs. -16.6 ± 3.0%, P < 0.01), and cardiac atrophy (LV mass index [mg/mm], 4.3 ± 0.1 vs. 3.6 ± 0.2, P < 0.01). Regional sheetlet angles were significantly lower in AIC, whereas helix angle and relative helicity remained unchanged. In AIC, fractional anisotropy was increased (0.12 ± 0.01 vs. 0.14 ± 0.02, P < 0.05). DOX-treated mice displayed higher planar and less spherical anisotropy (CPlanar 0.07 ± 0.01 vs. 0.09 ± 0.01, P < 0.01; CSpherical 0.89 ± 0.01 vs. 0.87 ± 0.02, P < 0.05). CPlanar and CSpherical yielded good discriminatory power to distinguish between mice with and without AIC (c-index 0.91 and 0.84, respectively, P for both < 0.05). AIC is associated with regional changes in sheetlet angle but no major abnormalities of global LV microarchitecture. The geometric shape of the diffusion tensor is altered in AIC. DTI may provide a new tool for myocardial characterization in patients with AIC, which warrants future clinical studies to evaluate its diagnostic utility.

An altered plasma lipidome-phenome network characterizes heart failure with preserved ejection fraction.

AIMS: Heart failure with preserved ejection fraction (HFpEF) is a multifactorial, multisystemic syndrome that involves alterations in lipid metabolism. This study aimed to test whether distinct plasma lipid profiles or lipid entities or both are associated with clinical and functional echocardiographic parameters in HFpEF. METHODS AND RESULTS: We examined the human plasma lipidome in HFpEF patients (n = 18) with left ventricular ejection fraction ≥50% and N-terminal pro-brain natriuretic peptide (NT-proBNP) >125 pg/mL and control subjects (n = 12) using mass spectrometry-based shotgun lipidomics. The cohort included 8 women and 22 men with average age of 67.8 ± 8.6 SD. The control and disease groups were not significantly different with respect to age, body mass index, systolic and diastolic blood pressure, and waist-to-hip ratio. The disease group experienced more fatigue (P < 0.001), had more often coronary artery disease (P = 0.04), and received more medications (beta-blockers, P < 0.001). The disease group had significantly different levels of HFpEF-relevant parameters, including NT-proBNP (P < 0.001), left ventricular mass index (P = 0.005), left atrial volume index (P = 0.001), and left ventricular filling index (P < 0.001), and lower left ventricular end-diastolic diameter (P = 0.014), with no difference in left ventricular ejection fraction. Significant differences in lipid profiles between HFpEF patients and controls could not be detected, including no significant differences in abundance of circulating lipids binned by carbon chain length or by double bonds, nor at the level of individual lipid species. However, there was a striking correlation between selected lipids with smoking status that was independent of disease status, as well as between specific lipids and hyperlipidaemia [with corresponding significance of either false discovery rate (FDR) <0.1 or FDR < 0.01]. In an exploratory network analysis of correlations, we observed significantly stronger correlations within the HFpEF group between individual lipids from the cholesterol ester and phosphatidylcholine (PC) classes and clinical/echocardiographic parameters such as left atrial volume index, left ventricular end-diastolic diameters, and heart rate (FDR < 0.1). In contrast, the control group showed significantly stronger negative correlations (FDR < 0.1) between individual species from the PC and sphingomyelin classes and left ventricular mass index or systolic blood pressure. CONCLUSIONS: We did not find significant direct associations between plasma lipidomic parameters and HFpEF and therefore could not conclude that any specific lipids are biomarkers of HFpEF. The validation in larger cohort is needed to confidently conclude the absence of first-order associations.

Finerenone Reduces Renal RORγt γδ T Cells and Protects against Cardiorenal Damage.

INTRODUCTION: Chronic activation of the mineralocorticoid receptor (MR) leads to pathological processes like inflammation and fibrosis during cardiorenal disease. Modulation of immunological processes in the heart or kidney may serve as a mechanistic and therapeutic interface in cardiorenal pathologies. In this study, we investigated anti-inflammatory/-fibrotic and immunological effects of the selective nonsteroidal MR antagonists finerenone (FIN) in the deoxycorticosterone acetate (DOCA)-salt model. METHODS: Male C57BL6/J mice were uninephrectomized and received a DOCA pellet implantation (2.4 mg/day) plus 0.9% NaCl in drinking water (DOCA-salt) or received a sham operation and were orally treated with FIN (10 mg/kg/day) or vehicle in a preventive study design. Five weeks after the procedure, blood pressure (BP), urinary albumin/creatinine ratio (UACR), glomerular and tubulointerstitial damage, echocardiographic cardiac function, as well as cardiac/renal inflammatory cell content by FACS analysis were assessed. RESULTS: BP was significantly reduced by FIN. FACS analysis revealed a notable immune response due to DOCA-salt exposure. Especially, infiltrating renal RORγt γδ-positive T cells were upregulated, which was significantly ameliorated by FIN treatment. This was accompanied by a significant reduction of UACR in FIN-treated mice. In the heart, FIN reduced DOCA-salt-induced cardiac hypertrophy, cardiac fibrosis and led to an improvement of the global longitudinal strain. Cardiac actions of FIN were not associated with a regulation of cardiac RORγt γδ-positive T cells. DISCUSSION/CONCLUSION: The present study shows cardiac and renal protective effects of FIN in a DOCA-salt model. The cardiorenal protection was accompanied by a reduction of renal RORγt γδ T cells. The observed actions of FIN may provide a potential mechanism of its efficacy recently observed in clinical trials.

Assessment of Myocardial Microstructure in a Murine Model of Obesity-Related Cardiac Dysfunction by Diffusion Tensor Magnetic Resonance Imaging at 7T.

Background: Obesity exerts multiple deleterious effects on the heart that may ultimately lead to cardiac failure. This study sought to characterize myocardial microstructure and function in an experimental model of obesity-related cardiac dysfunction. Methods: Male C57BL/6N mice were fed either a high-fat diet (HFD; 60 kcal% fat, n = 12) or standard control diet (9 kcal% fat, n = 10) for 15 weeks. At the end of the study period, cardiac function was assessed by ultra-high frequency echocardiography, and hearts were processed for further analyses. The three-dimensional myocardial microstructure was examined ex vivo at a spatial resolution of 100 × 100 × 100 µm3 by diffusion tensor magnetic resonance imaging (DT-MRI) at 7T. Myocardial deformation, diffusion metrics and fiber tract geometry were analyzed with respect to the different myocardial layers (subendocardium/subepicardium) and segments (base/mid-cavity/apex). Results were correlated with blood sample analyses, histopathology, and gene expression data. Results: HFD feeding induced significantly increased body weight combined with a pronounced accumulation of visceral fat (body weight 42.3 ± 5.7 vs. 31.5 ± 2.2 g, body weight change 73.7 ± 14.8 vs. 31.1 ± 6.6%, both P < 0.001). Obese mice showed signs of diastolic dysfunction, whereas left-ventricular ejection fraction and fractional shortening remained unchanged (E/e' 41.6 ± 16.6 vs. 24.8 ± 6.0, P < 0.01; isovolumic relaxation time 19 ± 4 vs. 14 ± 4 ms, P < 0.05). Additionally, global longitudinal strain was reduced in the HFD group (-15.1 ± 3.0 vs. -20.0 ± 4.6%, P = 0.01), which was mainly driven by an impairment in basal segments. However, histopathology and gene expression analyses revealed no myocardial fibrosis or differences in cardiomyocyte morphology. Mean diffusivity and eigenvalues of the diffusion tensor were lower in the basal subepicardium of obese mice as assessed by DT-MRI (P < 0.05). The three-dimensional fiber tract arrangement of the left ventricle (LV) remained preserved. Conclusion: Fifteen weeks of high-fat diet induced alterations in myocardial diffusion properties in mice, whereas no remodeling of the three-dimensional myofiber arrangement of the LV was observed. Obese mice showed reduced longitudinal strain and lower mean diffusivity predominantly in the left-ventricular base, and further investigation into the significance of this regional pattern is required.

Pharmacological inhibition of adipose tissue adipose triglyceride lipase by Atglistatin prevents catecholamine-induced myocardial damage.

AIMS: Heart failure (HF) is characterized by an overactivation of ß-adrenergic signalling that directly contributes to impairment of myocardial function. Moreover, ß-adrenergic overactivation induces adipose tissue lipolysis, which may further worsen the development of HF. Recently, we demonstrated that adipose tissue-specific deletion of adipose triglyceride lipase (ATGL) prevents pressure-mediated HF in mice. In this study, we investigated the cardioprotective effects of a new pharmacological inhibitor of ATGL, Atglistatin, predominantly targeting ATGL in adipose tissue, on catecholamine-induced cardiac damage. METHODS AND RESULTS: Male 129/Sv mice received repeated injections of isoproterenol (ISO, 25 mg/kg BW) to induce cardiac damage. Five days prior to ISO application, oral Atglistatin (2 mmol/kg diet) or control treatment was started. Two and twelve days after the last ISO injection cardiac function was analysed by echocardiography. The myocardial deformation was evaluated using speckle-tracking-technique. Twelve days after the last ISO injection, echocardiographic analysis revealed a markedly impaired global longitudinal strain, which was significantly improved by the application of Atglistatin. No changes in ejection fraction were observed. Further studies included histological-, WB-, and RT-qPCR-based analysis of cardiac tissue, followed by cell culture experiments and mass spectrometry-based lipidome analysis. ISO application induced subendocardial fibrosis and a profound pro-apoptotic cardiac response, as demonstrated using an apoptosis-specific gene expression-array. Atglistatin treatment led to a dramatic reduction of these pro-fibrotic and pro-apoptotic processes. We then identified a specific set of fatty acids (FAs) liberated from adipocytes under ISO stimulation (palmitic acid, palmitoleic acid, and oleic acid), which induced pro-apoptotic effects in cardiomyocytes. Atglistatin significantly blocked this adipocytic FA secretion. CONCLUSION: This study demonstrates cardioprotective effects of Atglistatin in a mouse model of catecholamine-induced cardiac damage/dysfunction, involving anti-apoptotic and anti-fibrotic actions. Notably, beneficial cardioprotective effects of Atglistatin are likely mediated by non-cardiac actions, supporting the concept that pharmacological targeting of adipose tissue may provide an effective way to treat cardiac dysfunction.

Wt1 haploinsufficiency induces browning of epididymal fat and alleviates metabolic dysfunction in mice on high-fat diet.

AIMS/HYPOTHESIS: Despite a similar fat storing function, visceral (intra-abdominal) white adipose tissue (WAT) is detrimental, whereas subcutaneous WAT is considered to protect against metabolic disease. Recent findings indicate that thermogenic genes, expressed in brown adipose tissue (BAT), can be induced primarily in subcutaneous WAT. Here, we investigate the hypothesis that the Wilms tumour gene product (WT1), which is expressed in intra-abdominal WAT but not in subcutaneous WAT and BAT, suppresses a thermogenic program in white fat cells. METHODS: Heterozygous Wt1 knockout mice and their wild-type littermates were examined in terms of thermogenic and adipocyte-selective gene expression. Glucose tolerance and hepatic lipid accumulation in these mice were assessed under normal chow and high-fat diet conditions. Pre-adipocytes isolated from the stromal vascular fraction of BAT were transduced with Wt1-expressing retrovirus, induced to differentiate and analysed for the expression of thermogenic and adipocyte-selective genes. RESULTS: Expression of the thermogenic genes Cpt1b and Tmem26 was enhanced and transcript levels of Ucp1 were on average more than tenfold higher in epididymal WAT of heterozygous Wt1 knockout mice compared with wild-type mice. Wt1 heterozygosity reduced epididymal WAT mass, improved whole-body glucose tolerance and alleviated severe hepatic steatosis upon diet-induced obesity in mice. Retroviral expression of WT1 in brown pre-adipocytes, which lack endogenous WT1, reduced mRNA levels of Ucp1, Ppargc1a, Cidea, Prdm16 and Cpt1b upon in vitro differentiation by 60-90%. WT1 knockdown in epididymal pre-adipocytes significantly lowered Aldh1a1 and Zfp423 transcripts, two key suppressors of the thermogenic program. Conversely, Aldh1a1 and Zfp423 mRNA levels were increased approximately five- and threefold, respectively, by retroviral expression of WT1 in brown pre-adipocytes. CONCLUSION/INTERPRETATION: WT1 functions as a white adipocyte determination factor in epididymal WAT by suppressing thermogenic genes. Reducing Wt1 expression in this and other intra-abdominal fat depots may represent a novel treatment strategy in metabolic disease.

Cardioprotective Effects of Palmitoleic Acid (C16:1n7) in a Mouse Model of Catecholamine-Induced Cardiac Damage Are Mediated by PPAR Activation.

Palmitoleic acid (C16:1n7) has been identified as a regulator of physiological cardiac hypertrophy. In the present study, we aimed to investigate the molecular pathways involved in C16:1n7 responses in primary murine cardiomyocytes (PCM) and a mouse model of isoproterenol (ISO)-induced cardiac damage. PCMs were stimulated with C16:1n7 or a vehicle. Afterwards, RNA sequencing was performed using an Illumina HiSeq sequencer. Confirmatory analysis was performed in PCMs and HL-1 cardiomyocytes. For an in vivo study, 129 sv mice were orally treated with a vehicle or C16:1n7 for 22 days. After 5 days of pre-treatment, the mice were injected with ISO (25 mg/kg/d s. c.) for 4 consecutive days. Cardiac phenotyping was performed using echocardiography. In total, 129 genes were differentially expressed in PCMs stimulated with C16:1n7, including Angiopoietin-like factor 4 (Angptl4) and Pyruvate Dehydrogenase Kinase 4 (Pdk4). Both Angptl4 and Pdk4 are proxisome proliferator-activated receptor α/δ (PPARα/δ) target genes. Our in vivo results indicated cardioprotective and anti-fibrotic effects of C16:1n7 application in mice. This was associated with the C16:1n7-dependent regulation of the cardiac PPAR-specific signaling pathways. In conclusion, our experiments demonstrated that C16:1n7 might have protective effects on cardiac fibrosis and inflammation. Our study may help to develop future lipid-based therapies for catecholamine-induced cardiac damage.

Depletion of cardiac cardiolipin synthase alters systolic and diastolic function.

Cardiolipin (CL) is a major cardiac mitochondrial phospholipid maintaining regular mitochondrial morphology and function in cardiomyocytes. Cardiac CL production includes its biosynthesis and a CL remodeling process. Here we studied the impact of CL biosynthesis and the enzyme cardiolipin synthase (CLS) on cardiac function. CLS and cardiac CL species were significantly downregulated in cardiomyocytes following catecholamine-induced cardiac damage in mice, accompanied by increased oxygen consumption rates, signs of oxidative stress, and mitochondrial uncoupling. RNAi-mediated cardiomyocyte-specific knockdown of CLS in Drosophila melanogaster resulted in marked cardiac dilatation, severe impairment of systolic performance, and slower diastolic filling velocity assessed by fluorescence-based heart imaging. Finally, we showed that CL72:8 is significantly decreased in cardiac samples from patients with heart failure with reduced ejection fraction (HFrEF). In summary, we identified CLS as a regulator of cardiac function. Considering the cardiac depletion of CL species in HFrEF, pharmacological targeting of CLS may be a promising therapeutic approach.

Adipose tissue-heart crosstalk as a novel target for treatment of cardiometabolic diseases.

Cardiometabolic disorders, such as diabetes, obesity, or metabolic syndrome, are often considered as key comorbidities, leading to the development of different forms of cardiovascular diseases such as heart failure or diabetic cardiomyopathy. Although the causal relationship between the pathophysiological status of white adipose tissue (WAT) and cardiac lipotoxicity is still elusive, elevated lipolytic rate in WAT has been demonstrated to participate in the overall augmentation of plasma lipid levels, as observed in most of the patients suffering from heart failure. In the present overview, we discuss current therapeutic approaches, as well as new treatment options targeting lipolysis and cardiac lipid metabolism in different forms of heart failure and diabetic cardiomyopathy.

Liver X Receptor Agonist AZ876 Induces Beneficial Endogenous Cardiac Lipid Reprogramming and Protects Against Isoproterenol-Induced Cardiac Damage.

Background It is known that dietary intake of polyunsaturated fatty acids may improve cardiac function. However, relatively high daily doses are required to achieve sufficient cardiac concentrations of beneficial omega-3 fatty acids. The liver X receptor (LXR) is a nuclear hormone receptor and a crucial regulator of lipid homeostasis in mammals. LXR activation has been shown to endogenously reprogram cellular lipid profiles toward increased polyunsaturated fatty acids levels. Here we studied whether LXR lipid reprogramming occurs in cardiac tissue and exerts cardioprotective actions. Methods and Results Male 129SV mice were treated with the LXR agonist AZ876 (20 µmol/kg per day) for 11 days. From day 6, the mice were injected with the nonselective ß-agonist isoproterenol for 4 consecutive days to induce diastolic dysfunction and subendocardial fibrosis while maintaining systolic function. Treatment with isoproterenol led to a marked impairment of global longitudinal strain and the E/e' ratio of transmitral flow to mitral annular velocity, which were both significantly improved by the LXR agonist. Histological examination showed a significant reduction in isoproterenol-induced subendocardial fibrosis by AZ876. Analysis of the cardiac lipid composition by liquid chromatography-high resolution mass spectrometry revealed a significant increase in cardiac polyunsaturated fatty acids levels and a significant reduction in saturated fatty acids by AZ876. Conclusions The present study provides evidence that the LXR agonist AZ876 prevents subendocardial damage, improves global longitudinal strain and E/e' in a mouse model of isoproterenol-induced cardiac damage, accompanied by an upregulation of cardiac polyunsaturated fatty acids levels. Cardiac LXR activation and beneficial endogenous cardiac lipid reprogramming may provide a new therapeutic strategy in cardiac disease with diastolic dysfunction.

Fat-body brummer lipase determines survival and cardiac function during starvation in Drosophila melanogaster.

The cross talk between adipose tissue and the heart has an increasing importance for cardiac function under physiological and pathological conditions. This study characterizes the role of fat body lipolysis for cardiac function in Drosophila melanogaster. Perturbation of the function of the key lipolytic enzyme, brummer (bmm), an ortholog of the mammalian ATGL (adipose triglyceride lipase) exclusively in the fly's fat body, protected the heart against starvation-induced dysfunction. We further provide evidence that this protection is caused by the preservation of glycerolipid stores, resulting in a starvation-resistant maintenance of energy supply and adequate cardiac ATP synthesis. Finally, we suggest that alterations of lipolysis are tightly coupled to lipogenic processes, participating in the preservation of lipid energy substrates during starvation. Thus, we identified the inhibition of adipose tissue lipolysis and subsequent energy preservation as a protective mechanism against cardiac dysfunction during catabolic stress.

Hypertrophy-Reduced Autophagy Causes Cardiac Dysfunction by Directly Impacting Cardiomyocyte Contractility.

Cardiac remodeling and contractile dysfunction are leading causes in hypertrophy-associated heart failure (HF), increasing with a population's rising age. A hallmark of aged and diseased hearts is the accumulation of modified proteins caused by an impaired autophagy-lysosomal-pathway. Although, autophagy inducer rapamycin has been described to exert cardioprotective effects, it remains to be shown whether these effects can be attributed to improved cardiomyocyte autophagy and contractility. In vivo hypertrophy was induced by transverse aortic constriction (TAC), with mice receiving daily rapamycin injections beginning six weeks after surgery for four weeks. Echocardiographic analysis demonstrated TAC-induced HF and protein analyses showed abundance of modified proteins in TAC-hearts after 10 weeks, both reduced by rapamycin. In vitro, cardiomyocyte hypertrophy was mimicked by endothelin 1 (ET-1) and autophagy manipulated by silencing Atg5 in neonatal cardiomyocytes. ET-1 and siAtg5 decreased Atg5-Atg12 and LC3-II, increased natriuretic peptides, and decreased amplitude and early phase of contraction in cardiomyocytes, the latter two evaluated using ImageJ macro Myocyter recently developed by us. ET-1 further decreased cell contractility in control but not in siAtg5 cells. In conclusion, ET-1 decreased autophagy and cardiomyocyte contractility, in line with siAtg5-treated cells and the results of TAC-mice demonstrating a crucial role for autophagy in cardiomyocyte contractility and cardiac performance.

Myocardial Infarction After High-Dose Catecholamine Application-A Case Report From an Experimental Imaging Study.

Although heart failure following myocardial infarction (MI) represents a major health burden, underlying microstructural and functional changes remain incompletely understood. Here, we report on a case of unexpected MI after treatment with the catecholamine isoproterenol in an experimental imaging study in mice using different state-of-the-art imaging modalities. The decline in cardiac function was documented by ultrahigh-frequency echocardiography and speckle-tracking analyses. Myocardial microstructure was studied ex vivo at a spatial resolution of 100 × 100 × 100 µm3 using diffusion tensor magnetic resonance imaging (DT-MRI) and histopathologic analyses. Two weeks after ISO treatment, the animal showed an apical aneurysm accompanied by reduced radial strain in corresponding segments and impaired global systolic function. DT-MRI revealed a loss of contractile fiber tracts together with a disarray of remaining fibers as corresponding microstructural correlates. This preclinical case report provides valuable insights into pathophysiology and morphologic-functional relations of heart failure following MI using emerging imaging technologies.

The Role of Adipose Triglyceride Lipase and Cytosolic Lipolysis in Cardiac Function and Heart Failure.

Heart failure is one of the leading causes of death worldwide. New therapeutic concepts are urgently required to lower the burden of heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF), the two major forms of heart failure. Lipolytic processes are induced during the development of heart failure and occur in adipose tissue and multiple organs, including the heart. Increasing evidence suggests that cellular lipolysis, in particular, adipose triglyceride lipase (ATGL) activity, has an important function in cardiac (patho)physiology. This review summarizes the crucial role of cellular lipolysis for normal cardiac function and for the development of HFrEF and HFpEF. We discuss the most relevant pre-clinical studies and elaborate on the cardiac consequences of non-myocardial and myocardial lipolysis modulation. Finally, we critically analyze the therapeutic importance of pharmacological ATGL inhibition as a potential treatment option for HFrEF and/or HFpEF in the future.

Obesity-related hypoxia via miR-128 decreases insulin-receptor expression in human and mouse adipose tissue promoting systemic insulin resistance.

BACKGROUND: Insulin resistance in visceral adipose tissue (VAT), skeletal muscle and liver is a prominent feature of most patients with obesity. How this association arises remains poorly understood. The objective of this study was to demonstrate that the decrease in insulin receptor (INSR) expression and insulin signaling in VAT from obese individuals is an early molecular manifestation that might play a crucial role in the cascade of events leading to systemic insulin resistance. METHODS: To clarify the role of INSR and insulin signaling in adipose tissue dysfunction in obesity, we first measured INSR expression in VAT samples from normal-weight subjects and patients with different degrees of obesity. We complemented these studies with experiments on high-fat diet (HFD)-induced obese mice, and in human and murine adipocyte cultures, in both normoxic and hypoxic conditions. FINDINGS: An inverse correlation was observed between increasing body mass index and decreasing INSR expression in VAT of obese humans. Our results indicate that VAT-specific downregulation of INSR is an early event in obesity-related adipose cell dysfunction, which increases systemic insulin resistance in both obese humans and mice. We also provide evidence that obesity-related hypoxia in VAT plays a determinant role in this scenario by decreasing INSR mRNA stability. This decreased stability is through the activation of a miRNA (miR-128) that downregulates INSR expression in adipocytes. INTERPRETATION: We present a novel pathogenic mechanism of reduced INSR expression and insulin signaling in adipocytes. Our data provide a new explanation linking obesity with systemic insulin resistance. FUNDING: This work was partly supported by a grant from Nutramed (PON 03PE000_78_1) and by the European Commission (FESR FSE 2014-2020 and Regione Calabria).

Serelaxin Improves Regional Myocardial Function in Experimental Heart Failure: An In Vivo Cardiac Magnetic Resonance Study.

Background Animal studies demonstrated that serelaxin lessens fibrosis in heart failure. This study assessed its effect on myocardial deformation using cardiac magnetic resonance and elucidated its relationship to gene regulation and histology in a mouse heart failure model. Methods and Results C57BL/6J mice were subjected to SHAM (n=4) or transverse aortic constriction (TAC). At week 10, TAC mice were randomized to receive either serelaxin (0.5 mg/kg per day; n=11) or vehicle (n=13) for 4 weeks. Cardiac magnetic resonance imaging was performed at baseline and repeated at the end of the study (week 14). Cine images were used to calculate left ventricular (LV) global longitudinal, circumferential, and radial strain. Hearts were examined for histology and gene expression. Compared with SHAM, mice 10 weeks after TAC showed increased LV mass with significant decreases in LV deformation parameters, indicating subclinical deterioration of myocardial function. At week 14, TAC mice given serelaxin demonstrated significant improvements in all LV strain parameters and no decrease in LV stroke volume and ejection fraction compared with TAC mice given vehicle. A significant positive correlation between global circumferential strain and the extent of myocardial fibrosis was found, and global circumferential strain correlated significantly with the expression of heart failure genes in serelaxin-treated mice. Conclusions Serelaxin improved cardiac magnetic resonance-derived myocardial deformation parameters as well as histomorphometric and gene expression findings in mice with heart failure. Cardiac magnetic resonance-derived myocardial mechanics correlate with histology and gene expression, stressing its utilization in myocardial remodeling.

Characterization of Myocardial Microstructure and Function in an Experimental Model of Isolated Subendocardial Damage.

Subendocardial damage is among the first cardiac manifestations of hypertension and is already present in asymptomatic disease states. Accordingly, markers of subendocardial impairment may facilitate early detection of cardiac damages and risk stratification under these conditions. This study aimed to investigate the impact of subendocardial damage on myocardial microstructure and function to elucidate early pathophysiologic processes and to identify corresponding diagnostic measures. Mice (n=38) were injected with isoproterenol to induce isolated subendocardial scarring or saline as corresponding control. Cardiac function and myocardial deformation were determined by high-frequency echocardiography. The cardiac stress response was assessed in a graded exercise test and during dobutamine stress echocardiography. Myocardial microstructure was studied ex vivo by 7 T diffusion tensor magnetic resonance imaging at a spatial resolution of 100×100×100 µm 3 . Results were correlated with histology and biomarker expression. Subendocardial fibrosis was accompanied by diastolic dysfunction, impaired longitudinal deformation (global peak longitudinal strain [LS]: -12.5±0.5% versus -15.6±0.5%; P<0.001) and elevated biomarker expression (ANP [atrial natriuretic peptide], Galectin-3, and ST2). Systolic function and cardiac stress response remained preserved. Diffusion tensor magnetic resonance imaging revealed a left-shift in helix angle towards lower values in isoproterenol-treated animals, which was mainly determined by subepicardial myofibers (mean helix angle: 2.2±0.8° versus 5.9±1.0°; P<0.01). Longitudinal strain and subepicardial helix angle were highly predictive for subendocardial fibrosis (sensitivity, 82%-92% and specificity, 89%-90%). The results indicate that circumscribed subendocardial damage alone can cause several hallmarks observed in cardiovascular high-risk patients. Microstructural remodeling under these conditions involves also remote regions, and corresponding changes in longitudinal strain and helix angle might serve as diagnostic markers.

Evaluation of a commercial multi-dimensional echocardiography technique for ventricular volumetry in small animals.

BACKGROUND: The assessment of ventricular volumes using conventional echocardiography methods is limited with regards to the need of geometrical assumptions. In the present study, we aimed to evaluate a novel commercial system for three-dimensional echocardiography (3DE) in preclinical models by direct comparison with conventional 1D- and 2D-echocardiography (1DE; 2DE) and the gold-standard technique magnetic resonance imaging (MRI). Further, we provide a standard operating protocol for image acquisition and analysis with 3DE. METHODS: 3DE was carried out using a 30 MHz center frequency transducer coupled to a Vevo®3100 Imaging System. We evaluated under different experimental conditions: 1) in vitro phantom measurements served as controlled setting in which boundaries were clearly delineated; 2) a validation cohort composed of healthy C57BL/6 J mice and New Zealand Obese (NZO) mice was used in order to validate 3DE against cardiac MRI; 3) a standard mouse model of pressure overload induced-heart failure was investigated to estimate the value of 3DE. RESULTS: First, in vitro volumetry revealed good agreement between 3DE assessed volumes and the MRI-assessed volumes. Second, cardiac volume determination with 3DE showed smaller mean differences compared to cardiac MRI than conventional 1DE and 2DE. Third, 3DE was suitable to detect reduced ejection fractions in heart failure mice. Fourth, inter- and intra-observer variability of 3DE showed good to excellent agreement regarding absolute volumes in healthy mice, whereas agreement rates for the relative metrics ejection fraction and stroke volume demonstrated good to moderate observer variabilities. CONCLUSIONS: 3DE provides a novel method for accurate volumetry in small animals without the need for spatial assumptions, demonstrating a technique for an improved analysis of ventricular function. Further validation work and highly standardized image analyses are required to increase reproducibility of this approach.

The cytoskeleton in 'couch potato-ism': Insights from a murine model of impaired actin dynamics.

Evidence for a critical pathophysiological role of aberrant cytoskeletal dynamics is being uncovered in a growing number of neuropsychiatric syndromes. A sedentary lifestyle as well as overt psychopathology is prevalent in patients with the metabolic syndrome. Using mice deficient in gelsolin (Gsn-/-), a crucial actin-severing protein, we here investigated reduced actin turnover as a potential common driver of metabolic disturbances, sedentary behavior, and an anxious/depressive phenotype. Gelsolin deficiency resulted in reduced lifespan. As compared to wildtype controls, Gsn-/- mice (~ 9 weeks) fed a high-fat diet (HFD) over a span of 12 weeks showed increased body weight gain, fat mass, hepatic steatosis, and adipocyte hypertrophy as well as a significantly reduced respiratory quotient. Moreover, increased rigidity of the actin cytoskeleton in mice on HFD induced mRNA expression of Acc1, Acc2, Fasn, and Lipe, key genes involved in fatty acid metabolism in the liver. Glucose tolerance and insulin sensitivity were worsened in Gsn-/- HFD relative to Gsn+/+ HFD mice. Hypertension in Gsn-/- mice was associated with reduced endothelial NO synthase (eNOS) mRNA expression and reduced eNOS protein trafficking to the plasma membrane. Furthermore, acetylcholine-induced cGMP production and relaxation of aortic rings were impaired by actin filament stabilization. Gsn-/- mice on HFD displayed reduced corticosterone concentrations and reduced energy expenditure as compared to Gsn+/+ HFD mice. Moreover, Gsn-/- HFD mice displayed an overall pattern of hypoactive and anxious/depressive-like behavior. In aggregate, our results demonstrate that impaired actin filament dynamics promote the development of key behavioral and physiological aspects of the metabolic syndrome.

Selective Mineralocorticoid Receptor Cofactor Modulation as Molecular Basis for Finerenone's Antifibrotic Activity.

Mineralocorticoid receptor antagonists (MRAs) reduce morbidity and mortality in chronic heart failure. Novel nonsteroidal MRAs are currently developed and need to be pharmacologically characterized in comparison to classical steroidal MRAs. A mouse model of cardiac fibrosis induced by short-term isoproterenol injection was used to compare the nonsteroidal MRA finerenone and the steroidal MRA eplerenone in equi-efficient systemic MR blocking dosages. Molecular mechanisms were studied in MR-expressing H9C2/MR+ cardiomyocytes and in MR transcriptional cofactor binding assays. Both MRAs significantly inhibited an isoproterenol-mediated increase of left ventricular mass. Isoproterenol-induced cardiac fibrosis and macrophage invasion were potently blocked by finerenone, whereas eplerenone had no significant effect. Speckle tracking echocardiography revealed a significant improvement of global longitudinal peak strain by finerenone, an effect less prominent with eplerenone. Antifibrotic actions of finerenone were accompanied by a significant inhibition of profibrotic cardiac TNX (tenascin-X) expression, a regulation absent with eplerenone. Finally, we show a higher potency/efficacy and inverse agonism of finerenone versus eplerenone in MR transcriptional cofactor binding assays indicating differential MR cofactor modulation by steroidal and nonsteroidal MRAs. This study demonstrates that the nonsteroidal MRA finerenone potently prevents cardiac fibrosis and improves strain parameters in mice. Cardiac antifibrotic actions of finerenone may result from the inhibition of profibrotic TNX gene expression mediated by differential MR cofactor binding. Selective MR cofactor modulation provides a molecular basis for distinct (pre)-clinical actions of nonsteroidal and steroidal MRAs.