Ventricular arrhythmias, a major cause of sudden cardiac death, are driven by Ca 2+ imbalance in cardiac myocytes, often linked to the overactivation of CaMKIIδ (Ca 2+ /calmodulin-dependent protein kinase II delta). As such, inhibiting CaMKIIδ represents a promising therapeutic strategy. Based on our previous finding that native secretoneurin (SN) is a weak CaMKIIδ inhibitor, we aimed to develop a more potent derivative of SN to effectively counter aberrant Ca 2+ handling and arrhythmia risk. Various regions of SN were tested for CaMKII binding, identifying the core region as the sequence with the strongest binding capacity. This region was subsequently optimised with two phenylalanine substitutions, resulting in the SN derivative SN-db-short. Structural homology modeling and ELISA-based assays revealed that SN-db-short bound both the substrate-binding (S-site) region of CaMKIIδ, in addition to the ATP-binding region, with 8-fold stronger binding compared to SN. Surface plasmon resonance experiments confirmed that SN-db-short exhibited a higher association rate and affinity for CaMKIIδ compared to SN. Consistent with only a partial calmodulin binding motif, SN-db-short showed no calmodulin binding, indicating selective CaMKIIδ inhibition. In functional studies, SN-db-short inhibited CaMKIIδ-mediated phosphorylation of ryanodine receptor 2 and appeared more effective than SN in reducing the incidence of Ca 2+ sparks and Ca 2+ waves. SN-db-short also more markedly inhibited CaMKIIδ phosphorylation of phospholamban, slowed Ca 2+ reuptake, and reduced the magnitude of Ca 2+ transients during isoproterenol stimulation. SN-db-short effectively inhibits CaMKIIδ and significantly counters aberrant Ca 2+ handling in cardiomyocytes. Thus, this optimised peptide holds therapeutic potential for reducing the risk of ventricular arrhythmias.

Syndecan-4 is a ubiquitously expressed transmembrane proteoglycan that links the extracellular matrix to intracellular protein networks. It is located at stress-sensing structures in cardiomyocytes, including costameres and Z-discs, and in male mice, it is involved in the hypertrophic response to cardiac pressure overload. We have recently found female syndecan-4 KO cardiomyocytes, without challenge, to be smaller in area. Smaller cardiomyocytes with elongation defects have been observed in animal models with β-parvin deficiency, where the loss of this mechano-sensor disrupts the guanine nucleotide exchange factor (GEF) β-PIX-GTPase Rac1 axis, which is essential for proper cell elongation. β-parvin, together with integrin-linked kinase (ILK) and particularly interesting new cysteine-histidine-rich protein (PINCH), constitutes the IPP complex (ILK-PINCH-parvin), which is part of the integrin consensus adhesome. Interestingly, in a previous large cardiac interactome study, we have identified β-parvin, as well as ILK, β-PIX, and Rac1 as potential syndecan-4 partners. To better understand the syndecan-4-β-parvin association, we mapped their interaction and investigated the effect of syndecan-4 ablation on the IPP complex, the β-parvin-β-PIX-Rac1 axis, and cardiomyocyte geometry in both females and males. Interestingly, genetic ablation of syndecan-4 resulted in shorter cardiomyocytes in females only. The syndecan-4-β-parvin interaction was mapped to accessible sequences within the N-terminal, linker, and CH2 domains of β-parvin and the unique variable C2 cytoplasmic region of syndecan-4. Syndecan-4 ablation resulted in lower levels of membrane-localized β-parvin in both sexes and sex-specific differences in its associated partners ILK and PINCH, suggesting that syndecan-4 is linked to integrin signaling through the IPP complex. Finally, Rac1, known for its involvement in cell size regulation, and some of its regulators, β-PIX, RhoGDIα, and the serine/threonine kinase PAK, showed sex-specific alterations following syndecan-4 ablation. Altogether, our data suggest that syndecan-4 binds directly to β-parvin and regulates cardiomyocyte length, the IPP complex, and the β-parvin-β-PIX-Rac1 in a sex-dependent manner. These findings highlight a sex-specific role for syndecan-4 in cardiomyocyte structure, offering new insight into the molecular basis for sex differences in cardiac biology.

BACKGROUND:High-throughput assays are required for novel biomarkers to have clinical potential. Secretoneurin (SN) is a candidate biomarker, and the performance of a new high-throughput SN assay is not known. METHODS:We measured SN concentrations with a prototype chemiluminescent immunoassay (CLIA) in 299 patients hospitalized with acute dyspnea. We compared the results with a CE-marked SN enzyme linked immunosorbent assay (ELISA). We adjudicated the cause of dyspnea as heart failure (HF) or non-HF, and we obtained information on all-cause mortality during follow-up. RESULTS:SN concentrations measured with CLIA and ELISA were closely correlated: rho = 0.81, P < 0.001. SN CLIA concentrations were higher in HF patients (n = 129) compared to patients with non-HF-related dyspnea (n = 170): median 51 (quartile 1-3 40-69) vs 41 (32-54) pmol/L, P < 0.001. The area under the curve (AUC) of SN CLIA to diagnose HF was 0.64 (95% CI, 0.58-0.71) and the AUC of N-terminal pro-B-type natriuretic peptide (NT-proBNP) was 0.85 (0.81-0.89). During median 818 days follow-up, 110 patients died (37%). There was a nonlinear association between SN CLIA concentrations and mortality with optimal cutpoint 53 pmol/L. SN CLIA concentrations >53 pmol/L were associated with mortality after adjusting for clinical variables and NT-proBNP and cardiac troponin T concentrations: hazard ratio 1.7 (95% CI, 1.1-2.7), AUC 0.67 (0.61-0.74). We found similar results for SN ELISA for diagnosis and prognosis with AUC 0.63 (0.57-0.70) for the prediction of mortality. CONCLUSION:The high-throughput SN CLIA correlates with the SN ELISA and provides independent prognostic information over established biomarkers in patients with acute dyspnea.

Abstract Inflammation through activation of caspase‐1, seems to play a role in pulmonary hypertension induced by alveolar hypoxia. Whether alveolar hypoxia induces caspase‐1‐mediated inflammation and influx of leukocytes in other organs than the lungs, is not known. Our aim was to explore sites of caspase‐1‐related inflammation in alveolar hypoxia. Wild type (WT) mice were exposed to environmental hypoxia or room‐air, and organs were analyzed. Right heart catheterization was performed after 14 days of alveolar hypoxia in WT mice and mice transplanted with WT or caspase‐1−/− bone marrow. Hypoxia induced leukocyte accumulation and increased caspase‐1 protein in the lungs, not in other organs. WT mice transplanted with WT or caspase‐1−/− bone marrow showed no difference in pulmonary leukocyte accumulation or development of pulmonary hypertension after alveolar hypoxia. Caspase‐1 and IL‐18 were detected in bronchial epithelium in WT mice, and hypoxia induced IL‐18 secretion from bronchial epithelial cells. IL‐18 stimulation generated IL‐6 mRNA in monocytes. Phosphorylated STAT3 was increased in hypoxic lungs, not in other organs. Alveolar hypoxia induces caspase‐1 activation and leukocyte accumulation specific to the lungs, not in other organs. Caspase‐1 activation and IL‐18 secretion from bronchial epithelial cells might initiate hypoxia‐induced inflammation, leading to pulmonary hypertension.

Mechanical load is a potent regulator of cardiac structure and function. Although high workload during heart failure is associated with disruption of cardiomyocyte t-tubules and Ca2+ homeostasis, it remains unclear whether changes in preload and afterload may promote adaptive t-tubule remodelling. We examined this issue by first investigating isolated effects of stepwise increases in load in cultured rat papillary muscles. Both preload and afterload increases produced a biphasic response, with the highest t-tubule densities observed at moderate loads, whereas excessively low and high loads resulted in low t-tubule levels. To determine the baseline position of the heart on this bell-shaped curve, mice were subjected to mildly elevated preload or afterload (1 week of aortic shunt or banding). Both interventions resulted in compensated cardiac function linked to increased t-tubule density, consistent with ascension up the rising limb of the curve. Similar t-tubule proliferation was observed in human patients with moderately increased preload or afterload (mitral valve regurgitation, aortic stenosis). T-tubule growth was associated with larger Ca2+ transients, linked to upregulation of L-type Ca2+ channels, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients advanced the heart down the declining limb of the t-tubule-load relationship. This bell-shaped relationship was lost in the absence of electrical stimulation, indicating a key role of systolic stress in controlling t-tubule plasticity. In conclusion, modest augmentation of workload promotes compensatory increases in t-tubule density and Ca2+ cycling, whereas this adaptation is reversed in overloaded hearts during heart failure progression. KEY POINTS: Excised papillary muscle experiments demonstrated a bell-shaped relationship between cardiomyocyte t-tubule density and workload (preload or afterload), which was only present when muscles were electrically stimulated. The in vivo heart at baseline is positioned on the rising phase of this curve because moderate increases in preload (mice with brief aortic shunt surgery, patients with mitral valve regurgitation) resulted in t-tubule growth. Moderate increases in afterload (mice and patients with mild aortic banding/stenosis) similarly increased t-tubule density. T-tubule proliferation was associated with larger Ca2+ transients, with upregulation of the L-type Ca2+ channel, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients placed the heart on the declining phase of the t-tubule-load relationship, promoting heart failure progression. The dependence of t-tubule structure on preload and afterload thus enables both compensatory and maladaptive remodelling, in rodents and humans.

AbstractAimsSacubitril/valsartan (Sac/Val) is used for treatment of heart failure. The effect of Sac/Val on regional dysfunction following myocardial infarction (MI) remains uncertain. This study aimed at understanding the effects of Sac/Val on regional function after MI.Methods and resultsMI or sham surgery was performed in Sprague–Dawley rats. Animals were randomized to treatment with Sac/Val, valsartan (Val) or vehicle (Veh). Magnetic resonance imaging was used to acquire left ventricular volumes and strain. Left ventricular tissue was obtained for wesern blotting, PCR and Masson's trichrome staining. Isolated cardiac fibroblasts were cultured with Veh, atrial natriuretic peptide (ANP), adrenomedullin (ADM) and sacubitrilat, and collagen expression assessed with droplet digital PCR.ResultsSac/Val reduced ventricular end‐diastolic volume by 18% compared with Veh, and preserved circumferential systolic strain in the zone proximal to infarction compared with sham after 42 days of treatment (peak strain ± SEM: sham: −0.19 ± 0.01%; Sac/Val: −0.14 ± 0.02%; Val: −0.10 ± 0.02%; Veh: −0.10 ± 0.02%). Masson's trichrome staining demonstrated lower fibrotic deposition in the intermediate zone with Sac/Val treatment than Veh (sham: 2.29 ± 0.17%; Sac/Val: 2.31 ± 0.27%; Val: 3.22 ± 0.60%; Veh: 4.14 ± 0.48%). The amounts of the pro‐apoptotic caspase 3 cleavage fragments p19/17 were 89% higher in Val than sham, with Sac/Val showing no significant increase compared with sham. Collagen expression in human fibroblast culture was lower in cells co‐treated with sacubitrilat and ANP, an effect not observed with sacubitrilat/ADM co‐treatment.ConclusionsSac/Val preserves in vivo myocardial function in the region most proximal to MI in rats and reduces left ventricular dilatation. These effects may be related to a reduction in both fibrosis and pro‐apoptotic signalling.

The transmembrane proteoglycan syndecan-4 is known to be involved in the hypertrophic response to pressure overload. Although multiple downstream signaling pathways have been found to be involved in this response in a syndecan-4-dependent manner, there are likely more signaling components involved. As part of a larger syndecan-4 interactome screening, we have previously identified MLP as a binding partner to the cytoplasmic tail of syndecan-4. Interestingly, many human MLP mutations have been found in patients with hypertrophic (HCM) and dilated cardiomyopathy (DCM). To gain deeper insight into the role of the syndecan-4–MLP interaction and its potential involvement in MLP-associated cardiomyopathy, we have here investigated the syndecan-4–MLP interaction in primary adult rat cardiomyocytes and the H9c2 cell line. The binding of syndecan-4 and MLP was analyzed in total lysates and subcellular fractions of primary adult rat cardiomyocytes, and baseline and differentiated H9c2 cells by immunoprecipitation. MLP and syndecan-4 localization were determined by confocal microscopy, and MLP oligomerization was determined by immunoblotting under native conditions. Syndecan-4–MLP binding, as well as MLP self-association, were also analyzed by ELISA and peptide arrays. Our results showed that MLP-WT and syndecan-4 co-localized in many subcellular compartments; however, their binding was only detected in nuclear-enriched fractions of isolated adult cardiomyocytes. In vitro, syndecan-4 bound to MLP at three sites, and this binding was reduced in some HCM-associated MLP mutations. While MLP and syndecan-4 also co-localized in many subcellular fractions of H9c2 cells, these proteins did not bind at baseline or after differentiation into cardiomyocyte-resembling cells. Independently of syndecan-4, mutated MLP proteins had an altered subcellular localization in H9c2 cells, compared to MLP-WT. The DCM- and HCM-associated MLP mutations, W4R, L44P, C58G, R64C, Y66C, K69R, G72R, and Q91L, affected the oligomerization of MLP with an increase in monomeric at the expense of trimeric and tetrameric recombinant MLP protein. Lastly, two crucial sites for MLP self-association were identified, which were reduced in most MLP mutations. Our data indicate that the syndecan-4–MLP interaction was present in nuclear-enriched fractions of isolated adult cardiomyocytes and that this interaction was disrupted by some HCM-associated MLP mutations. MLP mutations were also linked to changes in MLP oligomerization and self-association, which may be essential for its interaction with syndecan-4 and a critical molecular mechanism of MLP-associated cardiomyopathy.

Cardiac fibrosis is a central pathological feature in several cardiac diseases, but the underlying molecular players are insufficiently understood. The extracellular matrix proteoglycan versican is elevated in heart failure and suggested to be a target for treatment. However, the temporal expression and spatial distribution of versican and the versican cleavage fragment containing the neoepitope DPEAAE in cardiac fibrosis remains to be elucidated. In this study, we have examined versican during cardiac fibrosis development in a murine pressure overload model and in patients with cardiomyopathies. We found that versican, mainly the V1 isoform, was expressed immediately after induction of pressure overload, preceding collagen accumulation, and versican protein levels extended from the perivascular region into the cardiac interstitium. In addition, we found increased production of versican by collagen expressing fibroblasts, and that it was deposited extensively in the fibrotic extracellular matrix during pressure overload. In cardiac cell cultures, the expression of versican was induced by the pro-fibrotic transforming growth factor beta and mechanical stretch. Furthermore, we observed that the proteolytic cleavage of versican (DPEAAE fragment) increased in the late phase of fibrosis development during pressure overload. In patients with hypertrophic and dilated cardiomyopathies, we found elevated levels of versican and a positive correlation between versican and collagen mRNA in the heart, as well as increased cleavage of full-length protein. Taken together, the temporal expression profile and the spatial distribution of both the full-length versican and the DPEAAE fragment observed in this study indicates a role for versican in development of cardiac fibrosis.

Integrins are cell surface receptors expressed ubiquitously including on cardiac cells, which are critical for maintaining homeostasis in the cardiovascular system in health and function as key modulators of cardiac disease. They govern most facets of cardiac myocyte and fibroblast cellular function, act as important bidirectional mechanoreceptors, and interact with and respond to the extracellular matrix and its varied components. In this chapter, we focus on the growing body of knowledge identifying which of the integrin family members are expressed in the heart in health and disease, delineating the expression patterns of integrins in cardiac myocytes and fibroblasts, while highlighting important lessons learnt from cellular and animal models of cardiac disease. We will also highlight aspects of integrin biology obtained from humans, as available. Integrins lack intrinsic enzymatic activity to transduce mechanical to biochemical signaling, therefore, we will also discuss some of the identified binding and adapter proteins involved in integrin-mediated cell signaling relevant to the heart. We will present the currently understood therapeutic potential of select integrins in specific cardiac diseases, such as that following myocardial infarction or development of various cardiomyopathies. Most of this work has been largely unexplored in human cardiac disease. Finally, we highlight some of the pitfalls that have hampered our understanding of integrin and integrin-related protein targeting in heart disease and discuss the reemergence of integrins as attractive therapeutic targets.

Supplementary data for research article titled "Beneficial effects of exercise initiated prior to development of hypertrophic cardiomyopathy in genotype positive mice"

Circulating biomarkers reflecting cardiac inflammation are needed to improve the diagnostics and guide the treatment of heart failure patients. The cardiac production and shedding of the transmembrane proteoglycan syndecan-4 is upregulated by innate immunity signaling pathways. Here, we investigated the potential of syndecan-4 as a blood biomarker of cardiac inflammation. Serum syndecan-4 was measured in patients with (i) non-ischemic, non-valvular dilated cardiomyopathy (DCM), with (n = 71) or without (n = 318) chronic inflammation; (ii) acute myocarditis (n = 15), acute pericarditis (n = 3) or acute perimyocarditis (23) and (iii) acute myocardial infarction (MI) at day 0, 3 and 30 (n = 119). Syndecan-4 was investigated in cultured cardiac myocytes and fibroblasts (n = 6–12) treated with the pro-inflammatory cytokines interleukin (IL)-1β and its inhibitor IL-1 receptor antagonist (IL-1Ra), or tumor necrosis factor (TNF)α and its specific inhibitor infliximab, an antibody used in treatment of autoimmune diseases. The levels of serum syndecan-4 were comparable in all subgroups of patients with chronic or acute cardiomyopathy, independent of inflammation. Post-MI, syndecan-4 levels were increased at day 3 and 30 vs. day 0. IL-1Ra attenuated IL-1β-induced syndecan-4 production and shedding in vitro, while infliximab had no effect. In conclusion, syndecan-4 shedding from cardiac myocytes and fibroblasts was attenuated by immunomodulatory therapy. Although its circulating levels were increased post-MI, syndecan-4 did not reflect cardiac inflammatory status in patients with heart disease.

Abstract Aims Heart failure is a condition with high mortality rates, and there is a lack of therapies that directly target maladaptive changes in the extracellular matrix (ECM), such as fibrosis. We investigated whether the ECM enzyme known as A disintegrin and metalloprotease with thrombospondin motif (ADAMTS) 4 might serve as a therapeutic target in treatment of heart failure and cardiac fibrosis. Methods and results The effects of pharmacological ADAMTS4 inhibition on cardiac function and fibrosis were examined in rats exposed to cardiac pressure overload. Disease mechanisms affected by the treatment were identified based on changes in the myocardial transcriptome. Following aortic banding, rats receiving an ADAMTS inhibitor, with high inhibitory capacity for ADAMTS4, showed substantially better cardiac function than vehicle-treated rats, including ∼30% reduction in E/e′ and left atrial diameter, indicating an improvement in diastolic function. ADAMTS inhibition also resulted in a marked reduction in myocardial collagen content and a down-regulation of transforming growth factor (TGF)-β target genes. The mechanism for the beneficial effects of ADAMTS inhibition was further studied in cultured human cardiac fibroblasts producing mature ECM. ADAMTS4 caused a 50% increase in the TGF-β levels in the medium. Simultaneously, ADAMTS4 elicited a not previously known cleavage of TGF-β-binding proteins, i.e. latent-binding protein of TGF-β and extra domain A-fibronectin. These effects were abolished by the ADAMTS inhibitor. In failing human hearts, we observed a marked increase in ADAMTS4 expression and cleavage activity. Conclusion Inhibition of ADAMTS4 improves cardiac function and reduces collagen accumulation in rats with cardiac pressure overload, possibly through a not previously known cleavage of molecules that control TGF-β availability. Targeting ADAMTS4 may serve as a novel strategy in heart failure treatment, in particular, in heart failure with fibrosis and diastolic dysfunction.

Background: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca 2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3′-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. Methods: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca 2+ release was recorded using Fluo-4. Results: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca 2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca 2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. Conclusions: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

Introduction: N-terminal pro-B-type natriuretic peptide (NT-proBNP) and cardiac troponin T (cTnT) measurements are recommended in patients with acute dyspnea. We aimed to assess the prognostic merit of cTnT compared to NT-proBNP for 30-day readmission or death in patients hospitalized with acute dyspnea. Methods: We measured cTnT and NT-proBNP within 24 hours in 314 patients hospitalized with acute dyspnea and adjudicated the cause of the index admission. Time to first event of readmission or death ≤30 days after hospital discharge was recorded and cTnT and NT-proBNP measurements were compared head-to-head. Results: Five patients died during the index admission and were excluded from the study. Patients who died (7/309) or were readmitted (71/309) within 30 days had higher cTnT concentrations (median 29.1, Q1-Q3 18.2-73.9 ng/L) compared to patient who survived and were not readmitted (median 19.4, Q1-Q3 8.4-36.1 ng/L; p <0.001). There were no statistically significant differences in NT proBNP concentrations when stratifying patients according to readmission or death within 30 days: median 1540.6, Q1-Q3 424-5571 ng/L vs. median 984, Q1-Q3 201-3600 (p = 0.07). cTnT concentrations separated patients with poor and favorable outcomes, both for the total cohort and for patients with adjudicated heart failure (HF). cTnT concentrations were associated with readmission or death within 30 days after discharge, both in the total cohort (adjusted hazard ratio [aHR] 1.65, 95% CI 1.30-2.08) and in patients with HF (aHR 1.60, 95% CI 1.15-2.24). In contrast, NT-proBNP concentrations were not associated with prognosis in the total cohort (aHR 1.11, 95% CI 0.94-1.31) or in patients with adjudicated HF (aHR 1.03, 95% CI 0.78-1.37). In the subgroup analysis of HF patients, where patients with acute HF were grouped according to LVEF (HFpEF and HFrEF), cTnT and NT proBNP did not predict short-term outcomes for HFrEF, but cTnT predicted short-term outcomes in HFpEF patients. Conclusions: cTnT concentrations associate with post-discharge 30-day readmission or death in patients hospitalized with acute dyspnea, as well as in in patients adjudicated HF. The association of cTnT with outcomes in HF appears driven by strong associations of cTnT in patients with HFpEF.

Supplementary data for research article titled "Exercise training prior to manifestation of hypertrophic cardiomyopathy in mice attenuates expression of pro-fibrotic genes"

Heart failure is a major cause of morbidity and mortality worldwide, and can result from pressure overload, where cardiac remodelling is characterized by cardiomyocyte hypertrophy and death, fibrosis, and inflammation. In failing hearts, transforming growth factor (TGF)β drives cardiac fibroblast (CFB) to myofibroblast differentiation causing excessive extracellular matrix production and cardiac remodelling. New strategies to target pathological TGFβ signalling in heart failure are needed. Here we show that the secreted glycoprotein ADAMTSL3 regulates TGFβ in the heart. We found that Adamtsl3 knock-out mice develop exacerbated cardiac dysfunction and dilatation with increased mortality, and hearts show increased TGFβ activity and CFB activation after pressure overload by aortic banding. Further, ADAMTSL3 overexpression in cultured CFBs inhibits TGFβ signalling, myofibroblast differentiation and collagen synthesis, suggesting a cardioprotective role for ADAMTSL3 by regulating TGFβ activity and CFB phenotype. These results warrant future investigation of the potential beneficial effects of ADAMTSL3 in heart failure.

Abstract Background/Introduction Cardiac fibrosis is a central pathophysiological process accompanying most cardiac diseases including heart failure, and is a predictor of poor outcomes. The fibrotic process is mediated by activated cardiac fibroblasts (CFBs), so-called myofibroblasts, which produce excessive amounts of type I collagen and other ECM molecules. The ADAMTSL family of glycoproteins is upregulated in the cardiac extracellular matrix (ECM) during heart failure, but their function in the heart is unknown. Some ADAMTSL proteins are suggested to regulate the TGFβ, a major driver of myofibroblast activation and cardiac fibrosis. Purpose The purpose of this study was to examine the role of ADAMTSL3 in TGFβ-regulation and heart failure. Methods ADAMTSL3 expression was analysed in failing hearts of patients and mice. To study the functional role of ADAMTSL3 in the failing heart, an Adamtsl3 knock-out (L3-KO) mouse was generated, and WT and L3-KO littermates were subjected to experimental heart failure by aortic banding (AB), or sham surgery, for a total of six weeks. The mice were followed with echocardiography and MRI, and the left ventricles (LVs) were harvested one and six weeks after AB or sham surgery, with molecular analyses performed. To elucidate molecular functions of ADAMTSL3 in vitro, we overexpressed ADAMTSL3 in CFBs producing an extensive ECM. Results We determined that ADAMTSL3 was produced by CFBs and upregulated in failing hearts of patients and mice. The L3-KO mice had a normal cardiac phenotype at baseline, but upon increased LV afterload developed a dilated cardiomyopathy phenotype with increased LV dilation and reduced systolic function from one week post-AB. Furthermore, high mortality was observed in the L3-KO mice post-AB, with 60% vs. 96% survival of WTs over six weeks. At one week post-AB RNA sequencing of LVs revealed 233 differentially expressed genes in L3-KO vs. WT, with Col1a1 and Postn among the most upregulated, suggesting increased fibrosis and TGFβ signalling. Increased canonical TGFβ signalling was confirmed by increased SMAD2/3 phosphorylation and increased Lox expression in L3-KOs six weeks post-AB. In vitro, ADAMTSL3 overexpression in cultured CFBs resulted in reduced collagen synthesis and reduced expression of COL1A1, LOX, CTGF and POSTN, indicating anti-fibrotic properties. Furthermore, ADAMTSL3 inhibited the expression of ACTA2 and SPP1, reduced α-SMA protein by 25%, and reduced proliferation and CFB contraction, suggesting attenuated TGFβ signalling and inhibited myofibroblast differentiation. Conclusions ADAMTSL3 was upregulated in human and mouse heart failure, and served an anti-fibrotic and cardio-protective role in failing mouse hearts. Mechanistically, ADAMTSL3 was produced by CFBs and inhibited myofibroblast differentiation and collagen synthesis through TGFβ in cultured CFBs. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Research Council of Norway