Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling.

BACKGROUND: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. METHODS: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. RESULTS: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin ß1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of ß-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that ß1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with ß-blockers had a distinct cardiac ECM profile. CONCLUSIONS: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that ß-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Effect of Iron Isomaltoside on Skeletal Muscle Energetics in Patients With Chronic Heart Failure and Iron Deficiency.

BACKGROUND: Iron repletion augments exercise capacity in chronic heart failure (HF), but there is a lack of mechanistic data explaining how iron could augment exercise performance despite minimal changes in hemoglobin (Hb). Besides Hb, iron is an obligate component of mitochondrial enzymes that generate cellular energy in the form of adenosine triphosphate and phosphocreatine (PCr). Dynamic phosphorus magnetic resonance spectroscopy is a noninvasive tool that quantifies in vivo muscle energetics by measuring the kinetics of PCr recovery after exertion. We tested the hypothesis that intravenous iron repletion in chronic HF enhances skeletal muscle energetics as reflected by shorter PCr recovery half-times (PCr t1/2) on phosphorus magnetic resonance spectroscopy. METHODS: We enrolled 40 patients (50% anemic) with chronic HF, New York Heart Association class ≥II, left ventricular ejection fraction ≤45%, and iron deficiency (ferritin<100 µg/L or 100-300 µg/L with transferrin saturation <20%). Subjects underwent stratified (anemic versus nonanemic) randomization (1:1) to a single, double-blinded, total dose infusion of iron isomaltoside or saline placebo with end points reassessed early at 2 weeks posttreatment to minimize confounding from exercise adaptation. The primary end point was PCr t1/2 at 2 weeks. Secondary end points included ADP recovery half-time (ADP t1/2; energetic marker), iron status, symptoms, Hb, exercise capacity, and safety. RESULTS: In the total population, treatment groups were similar at baseline. At 2 weeks, iron isomaltoside improved PCr t1/2 (adjusted difference, -6.8 s; 95% CI, 11.5 to -2.1; P=0.006), ADP t1/2 (-5.3 s; 95% CI, -9.7 to -0.9; P=0.02), ferritin (304 ng/mL; 95% CI, 217-391; P<0.0001), transferrin saturation (6.8%; 95% CI, 2.7-10.8; P=0.002), New York Heart Association class (-0.23; 95% CI, -0.46 to -0.01; P=0.04), resting respiratory rate (-0.7 breaths/min; 95% CI, -1.2 to -0.2; P=0.009), and postexercise Borg dyspnea score (-2.0; 95% CI, -3.7 to -0.3; P=0.04), but not Hb (2.4 g/L; 95% CI, -3.5 to 8.4; P=0.41). Adverse events were similar between groups. In subgroup analyses, iron isomaltoside improved PCr t1/2 in anemic (-8.4 s; 95% CI, -16.7 to -0.2; P=0.04) and nonanemic (-5.2 s; 95% CI, -10.6 to 0.2; P=0.06) cohorts. CONCLUSIONS: In patients with chronic HF and iron deficiency, a total repletion dose of iron isomaltoside given at a single sitting is well tolerated and associated with faster skeletal muscle PCr t1/2 at 2 weeks, implying better mitochondrial function. Augmented skeletal muscle energetics might therefore be an important mechanism via which iron repletion confers benefits in chronic HF despite minimal Hb changes. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrialsregister.eu/ctr-search/trial/2012-005592-13/GB . Unique identifier: EudraCT 2012-005592-13.

Role of ADAMTS-5 in Aortic Dilatation and Extracellular Matrix Remodeling.

OBJECTIVE: Thoracic aortic aneurysm (TAA), a degenerative disease of the aortic wall, is accompanied by changes in the structure and composition of the aortic ECM (extracellular matrix). The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases has recently been implicated in TAA formation. This study aimed to investigate the contribution of ADAMTS-5 to TAA development. APPROACH AND RESULTS: A model of aortic dilatation by AngII (angiotensin II) infusion was adopted in mice lacking the catalytic domain of ADAMTS-5 (Adamts5Δcat). Adamts5Δcat mice showed an attenuated rise in blood pressure while displaying increased dilatation of the ascending aorta (AsAo). Interestingly, a proteomic comparison of the aortic ECM from AngII-treated wild-type and Adamts5Δcat mice revealed versican as the most upregulated ECM protein in Adamts5Δcat mice. This was accompanied by a marked reduction of ADAMTS-specific versican cleavage products (versikine) and a decrease of LRP1 (low-density lipoprotein-related protein 1). Silencing LRP1 expression in human aortic smooth muscle cells reduced the expression of ADAMTS5, attenuated the generation of versikine, but increased soluble ADAMTS-1. A similar increase in ADAMTS-1 was observed in aortas of AngII-treated Adamts5Δcat mice but was not sufficient to maintain versican processing and prevent aortic dilatation. CONCLUSIONS: Our results support the emerging role of ADAMTS proteases in TAA. ADAMTS-5 rather than ADAMTS-1 is the key protease for versican regulation in murine aortas. Further studies are needed to define the ECM substrates of the different ADAMTS proteases and their contribution to TAA formation.

Echocardiographic evaluation of diastolic function in mouse models of heart disease.

BACKGROUND: Mouse models of heart disease are extensively employed. The echocardiographic characterization of contractile function is usually focused on systolic function with fewer studies assessing diastolic function. Furthermore, the applicability of diverse echocardiographic parameters of diastolic function that are commonly used in humans has not been extensively evaluated in different pathophysiological models in mice. METHODS AND RESULTS: We used high resolution echocardiography to evaluate parameters of diastolic function in mouse models of chronic pressure overload (aortic constriction), volume overload (aorto-caval shunt), heart failure with preserved ejection fraction (HFpEF; DOCA-salt hypertension), and acute sarcoplasmic reticulum dysfunction induced by thapsigargin - all known to exhibit diastolic dysfunction. Left atrial area increased in all three chronic models while mitral E/A was difficult to quantify at high heart rates. Isovolumic relaxation time (IVRT) and Doppler E/E' increased significantly and the peak longitudinal strain rate during early filling (peak reverse longitudinal strain rate) decreased significantly after aortic constriction, with the changes being proportional to the magnitude of hypertrophy. In the HFpEF model, reverse longitudinal strain rate decreased significantly but changes in IVRT and E/E' were non-significant, consistent with less severe dysfunction. With volume overload, there was a significant increase in reverse longitudinal strain rate and decrease in IVRT, indicating a restrictive physiology. Acute thapsigargin treatment caused significant prolongation of IVRT and decrease in reverse longitudinal strain rate. CONCLUSION: These results indicate that the combined measurement of left atrial area plus reverse longitudinal strain rate and/or IVRT provide an excellent overall assessment of diastolic function in the diseased mouse heart, allowing distinction between different types of pathophysiology.

High-frequency speckle tracking echocardiography in the assessment of left ventricular function and remodeling after murine myocardial infarction.

The objectives of this study were to assess the feasibility and accuracy of high-frequency speckle tracking echocardiography (STE) in a murine model of myocardial infarction (MI). STE is used clinically to quantify global and regional cardiac function, but its application in mice is challenging because of the small cardiac size and rapid heart rates. A high-frequency micro-ultrasound system with STE (Visualsonics Vevo 2100) was compared against magnetic resonance imaging (MRI) for the assessment of global left ventricular (LV) size and function after murine MI. Animals subjected to coronary ligation (n = 46) or sham ligation (n = 27) were studied 4 wk postoperatively. Regional and global deformation were also assessed. STE-derived LV ejection fraction (EF) and mass correlated well with MRI indexes (r = 0.93, 0.77, respectively; P < 0.001), as did STE-derived mass with postmortem values (r = 0.80, P < 0.001). Higher STE-derived volumes correlated positively with MRI-derived infarct size (P < 0.01). Global strain parameters were significantly reduced after MI (all P < 0.001) and strongly correlated with LV mass and MRI-derived infarct size as promising surrogates for the extent of remodeling and infarction, respectively (both P < 0.05). Regional strain analyses showed that radial strain and strain rate were relatively preserved in anterior basal segments after MI compared with more apical segments (P < 0.001); however, longitudinal strain and strain rate were significantly impaired both basally and distally (P < 0.001). Strain-derived parameters of dyssynchrony were significantly increased in the MI group (P < 0.01). Analysis time for STE was 210 ± 45 s with acceptable inter- and intraobserver variability. In conclusion, high-frequency STE enables quantitative assessment of regional and global function in the remodeling murine LV after MI.

Lem2 is essential for cardiac development by maintaining nuclear integrity.

AIMS: Nuclear envelope integrity is essential for the compartmentalization of the nucleus and cytoplasm. Importantly, mutations in genes encoding nuclear envelope (NE) and associated proteins are the second highest cause of familial dilated cardiomyopathy. One such NE protein that causes cardiomyopathy in humans and affects mouse heart development is Lem2. However, its role in the heart remains poorly understood. METHODS AND RESULTS: We generated mice in which Lem2 was specifically ablated either in embryonic cardiomyocytes (Lem2 cKO) or in adult cardiomyocytes (Lem2 iCKO) and carried out detailed physiological, tissue, and cellular analyses. High-resolution episcopic microscopy was used for three-dimensional reconstructions and detailed morphological analyses. RNA-sequencing and immunofluorescence identified altered pathways and cellular phenotypes, and cardiomyocytes were isolated to interrogate nuclear integrity in more detail. In addition, echocardiography provided a physiological assessment of Lem2 iCKO adult mice. We found that Lem2 was essential for cardiac development, and hearts from Lem2 cKO mice were morphologically and transcriptionally underdeveloped. Lem2 cKO hearts displayed high levels of DNA damage, nuclear rupture, and apoptosis. Crucially, we found that these defects were driven by muscle contraction as they were ameliorated by inhibiting myosin contraction and L-type calcium channels. Conversely, reducing Lem2 levels to ∼45% in adult cardiomyocytes did not lead to overt cardiac dysfunction up to 18 months of age. CONCLUSIONS: Our data suggest that Lem2 is critical for integrity at the nascent NE in foetal hearts, and protects the nucleus from the mechanical forces of muscle contraction. In contrast, the adult heart is not detectably affected by partial Lem2 depletion, perhaps owing to a more established NE and increased adaptation to mechanical stress. Taken together, these data provide insights into mechanisms underlying cardiomyopathy in patients with mutations in Lem2 and cardio-laminopathies in general.

NADPH oxidase-4 promotes eccentric cardiac hypertrophy in response to volume overload.

AIMS: Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling. METHODS AND RESULTS: We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4-/-) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4-/- mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P < 0.05), with less LV hypertrophy at organ level (increase in LV weight/tibia length ratio of 25% vs. 43%, P < 0.01) and cellular level (cardiomyocyte cross-sectional area: 323 µm2 vs. 379 µm2, P < 0.01). LV ejection fraction, foetal gene expression, interstitial fibrosis, myocardial capillary density, and levels of myocyte apoptosis after Shunt were similar in the two genotypes. Myocardial phospho-Akt levels were increased after induction of Shunt in WT mice, whereas levels decreased in Nox4-/- mice (+29% vs. -21%, P < 0.05), associated with a higher level of phosphorylation of the S6 ribosomal protein (S6) and the eIF4E-binding protein 1 (4E-BP1) in WT compared to Nox4-/- mice. We identified that Akt activation in cardiac cells is augmented by Nox4 via a Src kinase-dependent inactivation of protein phosphatase 2A. CONCLUSION: Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.

Cardioprotective Effect of the Mitochondrial Unfolded Protein Response During Chronic Pressure Overload.

BACKGROUND: The mitochondrial unfolded protein response (UPRmt) is activated when misfolded proteins accumulate within mitochondria and leads to increased expression of mitochondrial chaperones and proteases to maintain protein quality and mitochondrial function. Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPRmt in the heart is unclear. OBJECTIVES: The purpose of this study was to: 1) identify conditions that activate the UPRmt in the heart; and 2) study the relationship among the UPRmt, mitochondrial function, and cardiac contractile function. METHODS: Cultured cardiac myocytes were subjected to different stresses in vitro. Mice were subjected to chronic pressure overload. Tissues and blood biomarkers were studied in patients with aortic stenosis. RESULTS: Diverse neurohumoral or mitochondrial stresses transiently induced the UPRmt in cultured cardiomyocytes. The UPRmt was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPRmt with nicotinamide riboside (which augments NAD+ pools) in cardiomyocytes in vitro or hearts in vivo significantly mitigated the reductions in mitochondrial oxygen consumption induced by these stresses. In mice subjected to pressure overload, nicotinamide riboside reduced cardiomyocyte death and contractile dysfunction. Myocardial tissue from patients with aortic stenosis also showed evidence of UPRmt activation, which correlated with reduced tissue cardiomyocyte death and fibrosis and lower plasma levels of biomarkers of cardiac damage (high-sensitivity troponin T) and dysfunction (N-terminal pro-B-type natriuretic peptide). CONCLUSIONS: These results identify the induction of the UPRmt in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPRmt ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.

Myocardial NADPH oxidase-4 regulates the physiological response to acute exercise.

Regular exercise has widespread health benefits. Fundamental to these beneficial effects is the ability of the heart to intermittently and substantially increase its performance without incurring damage, but the underlying homeostatic mechanisms are unclear. We identify the ROS-generating NADPH oxidase-4 (Nox4) as an essential regulator of exercise performance in mice. Myocardial Nox4 levels increase during acute exercise and trigger activation of the transcription factor Nrf2, with the induction of multiple endogenous antioxidants. Cardiomyocyte-specific Nox4-deficient (csNox4KO) mice display a loss of exercise-induced Nrf2 activation, cardiac oxidative stress and reduced exercise performance. Cardiomyocyte-specific Nrf2-deficient (csNrf2KO) mice exhibit similar compromised exercise capacity, with mitochondrial and cardiac dysfunction. Supplementation with an Nrf2 activator or a mitochondria-targeted antioxidant effectively restores cardiac performance and exercise capacity in csNox4KO and csNrf2KO mice respectively. The Nox4/Nrf2 axis therefore drives a hormetic response that is required for optimal cardiac mitochondrial and contractile function during physiological exercise.

Both cardiomyocyte and endothelial cell Nox4 mediate protection against hemodynamic overload-induced remodelling.

AIMS: NADPH oxidase-4 (Nox4) is an important reactive oxygen species (ROS) source that is upregulated in the haemodynamically overloaded heart. Our previous studies using global Nox4 knockout (Nox4KO) mice demonstrated a protective role of Nox4 during chronic abdominal aortic banding, involving a paracrine enhancement of myocardial capillary density. However, other authors who studied cardiac-specific Nox4KO mice reported detrimental effects of Nox4 in response to transverse aortic constriction (TAC). It has been speculated that these divergent results are due to cell-specific actions of Nox4 (i.e. cardiomyocyte Nox4 detrimental but endothelial Nox4 beneficial) and/or differences in the model of pressure overload (i.e. abdominal banding vs. TAC). This study aimed to (i) investigate whether the effects of Nox4 on pressure overload-induced cardiac remodelling vary according to the pressure overload model and (ii) compare the roles of cardiomyocyte vs. endothelial cell Nox4. METHODS AND RESULTS: Global Nox4KO mice subjected to TAC developed worse cardiac remodelling and contractile dysfunction than wild-type littermates, consistent with our previous results with abdominal aortic banding. Next, we generated inducible cardiomyocyte-specific Nox4 KO mice (Cardio-Nox4KO) and endothelial-specific Nox4 KO mice (Endo-Nox4KO) and studied their responses to pressure overload. Both Cardio-Nox4KO and Endo-Nox4KO developed worse pressure overload-induced cardiac remodelling and dysfunction than wild-type littermates, associated with significant decrease in protein levels of HIF1α and VEGF and impairment of myocardial capillarization. CONCLUSIONS: Cardiomyocyte as well as endothelial cell Nox4 contributes to protection against chronic hemodynamic overload-induced cardiac remodelling, at least in part through common effects on myocardial capillary density.

Validation of Transesophageal Echocardiographic In Vitro Measurements for Bioprosthetic Aortic Valves: Implications for Percutaneous Valve-in-Valve Therapy.

BACKGROUND: Percutaneous valve-in-valve therapy has become an important treatment option for failing bioprosthetic heart valves. Accurate assessment of valve internal diameter (ID) is essential for effective and safe treatment. These data may not be available in an individual patient, or the manufacturer-supplied dimensions may be incorrect because they do not allow for the space occupied by valve leaflet material. METHODS: In total, 2,332 two-dimensional and three-dimensional transesophageal echocardiographic in vitro measurements were performed using both Philips iE33 and GE Vivid E9 systems with a range of system settings on 53 bioprosthetic valves in all available sizes. Two-dimensional echocardiographic ID measurements were made in two orthogonal planes at the level of the sewing ring, and similar three-dimensional measurements were generated from multiplane reconstructions. They were compared with both manufacturer-supplied valve ID (MID) and the true ID (TID) measured with Hegar dilators. RESULTS: Both the iE33 and the Vivid 9 provided comparable valve ID measurements. TID was statistically significantly smaller than MID (P < .001). All echocardiographic measurements were closer to TID than to MID. Two-dimensional measurements were closest to TID because of higher spatial resolution. CONCLUSIONS: Transesophageal echocardiographic valve ID measurements compare well with TID, which is overestimated by MID. These findings have potentially important implications for valve-in-valve procedures because an inaccurate measurement of TID might lead to the wrong choice of implanted valve.

Pocket-size imaging device: effectiveness for ward-based transthoracic studies.

AIMS: Pocket-size imaging devices (PSID) are now available; their potential role in a hospital environment has been investigated but still remains undefined. METHODS AND RESULTS: We evaluated the effectiveness of PSID in 92 patients referred for bedside transthoracic echocardiogram (TTE). Patients were included where there was a focused clinical question: quantification of left ventricular function (LVF); presence of regional wall motion abnormalities (RWMA); evidence of pericardial effusion, exclusion of significant valve pathology. Each patient underwent an echocardiography evaluation using PSID and TTE. In 83 patients [k = 90%, 95% CI (82.2-95.4)], it was possible to answer the clinical question by PSID examination alone. There was agreement between the findings of PSID and TTE in 86 cases [79%; k = 47%, 95% CI (12.8-82.0)], in three cases, the clinical question was not answered by both modalities. When the clinical question was focused on LVF, the agreement was excellent [k = 96%, 95% CI (95.3-97.9)], as was the agreement in the detection of RWMA [k = 94.57%, 95% CI (82.4-95.1)]. There was also good concordance in the detection of valve pathology and pericardial effusion. Using PSID, the reduction in the scanning and reporting time was 66%. The cost-effectiveness analysis produced very favourable results: with PSE, we obtained an overall cost saving per scan of 76%, compared with TTE. CONCLUSION: This study demonstrates that PSID can provide a valuable alternative to TTE in the presence of focused clinical questions and can provide an efficient way of delivering a ward-based transthoracic echo service.