Table 1 Diagnostic Choices and Their Potential Treatment Focuses on of Specific DCM Causes DCM; p38 signaling in DCM; gene modification in sufferers with truncating variantsToxic (alcoholic beverages/medication, cardiotoxic chemotherapy)Complete background of toxin publicity; probability for urine toxicology screenDisease onset during/after toxin exposure; regression or resolution after withholdingNo specific focuses on: withholding or reducing exposure; usage of cardioprotective realtors in anthracycline toxicity (dexrazoxane)InflammationEMB, bloodstream sampling (eg, sIL2\R, CRP, galectin\3), CMRImmune cell infiltration in EMB, elevated T2\signal, elevated CRP/ESRPro\inflammatory pathways (eg IL\1)Autoimmune diseaseEMB, blood sampling, imaging, autoantibody display, presence of extracardiac featuresPresence of swelling and positive autoantibody titersAuto\antibodies (eg, (1)\AABs)ViralEMBCardiotropic disease presence having a viral weight 500?copies/g DNAVirus and subsequent cardiac inflammationElectrical12\lead ECG, ambulatory ECG monitoring 10?000 to 25?000?PVCs/d; (supra)ventricular tachycardiaAbnormal electrical pathwaysPeripartum(history of) PregnancyDisease onset during pregnancy up to 6?mo postpartumCleaved 16?kDa N\terminal fragment of prolactinCardiac metabolismBlood, cells and/or urine metabolomicsElevated acylcarnitines, upsurge in ketone bodiesMultiple strategies possible interfering using the metabolic substrate change (mainly involving mitochondrial pathways)Cardiac fibrosisEMB, CMR, bloodIncreased CFV; midmyocardial LE; increased fibrosis blood markersRAAS\pathway; angiotensin II\galectin\3\interleukin\6 axis; matricellular proteins; syndecan\4\osteopontin\lysyl oxidase\like axis Open in another window AAB indicates autoantibodies; CFV, collagen small fraction quantity; CMR, cardiac magnetic resonance; CRP, C\reactive proteins; DCM, dilated cardiomyopathy; EMB, endomyocardial biopsy; ESR, erythrocyte sedimentation rate; ICD, implantable cardiac defibrillator; LE, late enhancement; PVC, premature ventricular complicated; RAAS, renin\angiotensin\aldosterone program; WES, entire exome sequencing. Open in a separate window Figure 2 Complete diagnostic workup of a dilated cardiomyopathy patient to characterize the cardiac function and underlying cause. CRP shows C\reactive proteins; sIL2\R, soluble interleukin 2 receptor. PRESENT STATE of Guide\Driven Therapies The existing treatment of DCM patients will not change from general HF management, with the pharmacological cornerstone consisting of \blockers, RAS (renin\angiotensin system) inhibitors, aldosterone antagonists, and diuretics.17 One of the most recent guideline\changing breakthroughs in general HF treatment was the introduction from the angiotensin receptor\neprilysin inhibitor sacubitril\valsartan.18 The major PARADIGM (Prospective Comparison of ARNI with ACEI to Determine Effect on Global Mortality and Morbidity in Heart Failure) trial showed a risk reduced amount of loss of life and HF hospitalization in chronic HF sufferers treated with sacubitril\valsartan rather than enalapril. About 60% of the included patients had an ischemic cause, making the drug still fairly brand-new in neuro-scientific DCM. The results from the PARADIGM trial have already been analyzed hoc in a number of subgroups post.19 A chance remains to execute an additional post hoc analysis in only nonischemic HF patients to evaluate its effect in this subgroup. Targeting the Root Reason behind DCM: Determining Upstream Causes In DCM it might be the target to initiate therapies targeting important molecular processes driven by upstream causes. Knowledge regarding causes of DCM has expanded within the last few years significantly. The increasing variety of sufferers in registries and merging international databases provides us with important information regarding medical demonstration and prognosis of specific causes. Current recommendations do not include treatment strategies directed at key molecular processes driven by upstream causes, because evidence is mostly coming from single\middle pilot and retrospective cohort research (Desk?2). Is the time to make the next step toward multicenter Now, randomized tests (Desk?3). Table 2 Overview of All scholarly studies Investigating Trigger\Directed Remedies in Dilated Cardiomyopathy Individuals DCM patients Unravel molecular outcomes of particular gene mutations Gene correction therapies towards a scientific application InflammationNo guide\directed therapy; although there is certainly proof from retrospective research showing reap the benefits of immunosuppressionPhase 2B RCT in acute myocarditis using anakinra vs standard care Multicenter RCT using immunosuppressive therapy in inflammatory DCM Auto\immunityNo guideline\directed therapy; although there is usually evidence showing benefit from immunoadsorption Multicenter RCT using immunoadsorption in DCM with cardiotoxic autoantibodies ViralNo guideline\directed therapy; although there are retrospective research and case reviews showing reap the benefits of IVIg Stage 3 RCT using IVIg for chronic PVB19\related DCM RCT using particular antiviral therapies (val/\ganciclovir) Multicenter RCT for IVIg if stage 3 trial is positive ToxicNo guideline\directed therapy; withholding or reducing exposure has been shown to be the most effective; in some cases, cardioprotective compounds for anthracycline toxicity are suggested.Stage 1 RCT using MPCs in anthracycline\induced DCM Unravel molecular adjustments in cocaine\induced DCM Stage 2 RCT using stem cell therapy in cardiotoxic chemotherapy\induced DCM Specify the timing, dosage, and duration of prophylactic therapy to avoid HF onset in those sufferers getting cardiotoxic chemotherapy in danger ElectricalEarly treatment of electric disturbance (AF, ablation; still left bundle branch stop, CRTD) Better understanding in the pathomechanisms from the interplay FRAX486 between HF and electric disruptions to detect their causal associations and better stratify individuals who will benefit from (device) therapy PeripartumNo guideline\directed therapy; although 1 potential study showed reap the benefits of bromocriptine A placebo\controlled research with bromocriptine to assess basic safety and efficiency More data regarding long\term outcome of PPCM after bromocriptine use Downstream processesBiomechanical defectsNo guideline\directed therapyPhase 1 and stage 2 RCT using MYK\491 in DCM Follow\up research predicated on outcomes of current stage 1 and 2 RCT Investigate the function of omecamtiv in (genetic) DCM Myocardial damageNo guideline\directed therapy; although multiple research investigated the benefit from stem cell therapy Stage 3 RCT merging BMC and G\CSF in DCM seeing that follow\up over the REGENERATE\DCM Cardiac metabolismNo guideline\directed therapy; although multiple substances have been looked into showing benefit Even more data regarding lengthy\term outcome of DCM after trimetazidine Investigating extracardiac ramifications of perhexiline use Cardiac fibrosisNo guideline\directed therapyRCT using ICD or ILR implantation in DCM individuals with LGE about CMR Particular antifibrotic medication personalized to arrhythmic reversibility or burden of fibrosis Exploring the role of novel antifibrotic therapies with regard to cardiac fibrosis (ie, vanticumab, simtuzumab) Open in a separate window AF indicates atrial fibrillation; BMC, bone tissue marrow\produced stem cells; CMR, cardiovascular magnetic resonance; CRTD, cardiac resynchronization therapy gadget; DCM, dilated cardiomyopathy; G\CSF, granulocyte colony\stimulating element; HF, heart failing; ICD, implantable cardioverter\defibrillator; ILR, implantable loop recorder; IVIg, intravenous immunoglobulin; LGE, past due gadolinium improvement; MPC, mesenchymal progenitor cells; PPCM, peripartum cardiomyopathy; PVB19, parvovirus B19; RCT, randomized managed trial. Genetic Causes Inheritance of a pathogenic gene variant is currently considered an irreversible cause and clinical management is dependant on the increasing genotypeCphenotype understanding. To time, the monogenetic dogma is usually applied in clinical practice, whereas genetic DCM is mainly considered an autosomal dominant disease where 1 gene variant plays a part in the DCM phenotype. Nevertheless, increasing insights claim that DCM, besides being truly a monogenic disease, is potentially a polygenic, complex disease involving the conversation among multiple gene variants. Whether this complex disease model shall result in therapy assistance is yet to become discovered. Currently, led therapy in the field of (mono)genetics can roughly be split into 3 different strategies. The first strategy is dependant on related to the precise gene mutation, based on current genotypeCphenotype knowledge. The best example is definitely a lower threshold for cardiac defibrillator implantation in sufferers using a pathogenic lamin A/C (mutations.46 That is likely due to the result of additional environmental factors had a need to result in the phenotype on top of the genotype, creating the concept of susceptibility genes.2, 6, 14 This is relevant for truncating mutations especially, which will be the most prevalent among genetic causes for DCM. Another approach is to of a particular gene mutation. Many cardiomyopathy\related genes and their following proteins have specific functions in the cardiomyocyte. Loss (or gain) of function of some of these proteins will result in intracellular changes from the sign transduction for which the cardiomyocyte tries to adapt. These molecular changes have been well investigated in gene mutation, which is the first research involving genotype\particular therapy. Using the increasing possibilities of transcriptomics and proteomics, chances are how the modified sign transduction of additional gene mutations will be unraveled. Truncating mutations are a good example, as proof is certainly accumulating relating to adjustments in the cardiac fat burning capacity and energy homeostasis.49 The increase in the energy metabolism could be a mechanism of metabolic compensation for the sarcomere insufficiency.50 This transformation in metabolism linked to truncating mutations could be a valid downstream focus on for intervention to prevent disease onset toward DCM. The ultimate therapy is to in the individual patient. Several strategies are under analysis to do this healing goal such as (1) CRISPR/Cas9, which can target specific single mutations; (2) exon skipping, targeting all mutations within one or two 2 exons and their linked introns; and (3) gene substitute concentrating on all mutations simultaneously by gene transfer of the full\size cDNA.51 Since its finding in 2013, CRISPR/Cas9 is now learning to be a dear tool to research pathogenicity of gene mutations, create experimental models, and to develop a genome\editing therapy for particular gene mutations such as for example are the renowned, and exon missing in individual\specific cardiomyocytes derived from induced pluripotent stem cells could be rescued, stopping defective myofibril stability and assembly.54 Also, exon missing prevented the introduction of DCM and improved contractile efficiency in has been proven.51 Gene replacement continues to be realized by transducing the cells with AAV (adeno\associated virus).55 This successfully increased the expression of transcripts and suppressed the condition phenotype. Although most of the genome editing offers been proven to become feasible and encouraging in cells and mice, there are still many questions that need to become replied before progressing to individual trials with the 3 techniques mentioned. These include the basic safety of viral delivery and providing the vector to the proper place in the proper dose, which will be challenging. Gene alternative therapy depends on AAV with a high cardiac tropism and a cardiac\particular promotor, which is normally AAV9 in mouse versions. However, the very best AAV serotype for the human heart must be established still. non-specific cardiac tropism can result in off\target effects, solid host immunogenicity against the virus, insufficient AAV potency, and effectiveness and imperfect incorporation.56 Interestingly, intracoronary infusion of AAV1\SERCA2a in 123 individuals with HF with minimal ejection fraction had not been associated with improvement in clinical outcome.57 Despite the failure of the scholarly research, these findings should stimulate further research into the use of gene therapy to treat HF patients. Inflammatory Triggers So far, 3 randomized tests have examined the result of immunosuppressive therapies in DCM.20, 21, 58 The two 2 largest of the trials assessed the effect of prednisone and azathioprine. However, they were conducted before the period of quantitative immunohistochemistry for the evaluation of infiltrates, using hematoxylin and eosin staining and the Dallas criteria solely.20, 58, 59 Furthermore, molecular evaluation for viral existence in myocardial biopsies had not been available. Both trials showed no beneficial effect on mortality, where the IMAC (Intervention in Myocarditis and Severe Cardiomyopathy) trial also didn’t show helpful results on cardiac function. Interestingly, Frustaci et?al demonstrated improved and sustained cardiac function in DCM individuals treated with immunosuppressive therapy in addition to HF therapy in comparison with those that received regular HF treatment by itself. The importance of discrimination between disease\positive and \bad individuals was exemplified by a post hoc stratification of sufferers treated with prednisone and azathioprine, showing beneficial results in the virus\negative group predominantly.60 Subsequently, a FRAX486 prospective research was done using immunosuppressive therapy in 85 individuals with aggressive cardiac swelling, but the absence of virus revealed a significantly improved cardiac function at 6?months in the immunosuppression group as compared with regular HF therapy only. Aside from the potential helpful results on cardiac function, long\term transplantation\free survival can also be improved applying this immunosuppressive routine. 22 Predicated on these outcomes, the most recent European Society of Cardiology recommendations declare that immunosuppression could be considered in contamination\harmful myocarditis refractory to regular therapy in sufferers without contraindications to immunosuppression.61, 62 On the other hand, these recommendations are not advocated by the American Heart Association guidelines.63 To prove potential great things about immunosuppression within this subset of patients definitively, a multicenter trial was initiated but terminated due to a low inclusion rate (“type”:”clinical-trial”,”attrs”:”text”:”NCT01877746″,”term_id”:”NCT01877746″NCT01877746). Therefore, confirmation of a randomized, prospective, placebo\controlled multicenter trial will be necessary before a consensus is certainly reached in suggestions. Recently, the role of IL (interleukin)\1 blockade, namely, anakinra, has elevated interest because clinical pilot research in inflammatory DCM demonstrated beneficial effects in hemodynamics, inflammation, and clinical overall performance.23, 24 Anakinra is a nonglycosylated protein that differs from your sequence of the local IL\1 receptor antagonist by 1 methionine put into its N\terminus. The IL\1 category of ligands and receptors is the main cytokine family members connected with severe and chronic swelling. Recently, 30 patients with severe decompensated HF and raised C\reactive protein levels were randomized to either receive anakinra or coordinating placebo.23 Anakinra reduced C\reactive proteins by 61% versus baseline, weighed against a 6% reduction among sufferers receiving placebo. Furthermore, anakinra was associated with a greater recovery in LVEF compared with placebo. No significant distinctions between treatment groupings in the original amount of stay or total hospital days during the 14?times were observed. Predicated on these advantageous results, security and efficacy of anakinra was evaluated in the REDHART Trial (Recently Decompensated Heart Failing Anakinra Response Trial), regarding 60 sufferers with lately decompensated heart failure and systolic dysfunction. Anakinra treatment decreases serum C\reactive proteins levels within a suffered manner and increases peak VO2. However, IL\1B blockade does not improve cardiac function or improve prognosis in terms of loss of life or HF hospitalization at 24?weeks as compared with placebo.24 However, the study was not powered to detect distinctions in outcome and really should be utilized as an estimation of effect size to design appropriately powered studies. Moreover, the ACTION Study Group in France initiated a Phase 2B randomized controlled trial in acute myocarditis patients using anakinra versus standard care (“type”:”clinical-trial”,”attrs”:”text message”:”NCT03018834″,”term_id”:”NCT03018834″NCT03018834). The analysis can be approximated to be completed in 2021. Blockade of IL\1B seems to be a guaranteeing therapy focus on in HF, as a recently available subgroup evaluation using placebo\controlled data of the CANTOS (Canakinumab Anti\Inflammatory Thrombosis Outcome Study) trial in which canakinumab (a monoclonal antibody against IL\1B) demonstrated a dosage\dependent reduction in HF hospitalization and HF\related mortality in 2173 patients with HF at baseline.64 Hence, 1 of the inclusion requirements for the CANTOS trial was a baseline C\reactive proteins 2?mg/L, therefore creating a selected population with signs of elevated systemic inflammation who benefitted from canakinumab treatment. Autoimmunity The concept of immunoadsorption involves removing cardiotoxic autoantibodies, as well as cytokines through the circulatory system, because they may cause damage to the myocardium.27, 65 In order to avoid infections, the adsorbed immunoglobulins (namely, IgG, IgA, and IgM) are replaced using administration of 0.5?g/kg body weight polyclonal IgG after treatment. Although studies are fairly little, an improvement of LV function is usually reported in 60% of sufferers treated.28 Using gene expression analysis in the myocardial tissue, responders and nonresponders could be potentially distinguished before treatment.66 Although these results appear attractive, confirmation with a randomized treatment trial is necessary. Viral Triggers Included in these are antiviral therapies targeting cardiotropic viruses such as herpes simplex virus 1 and 2, cytomegalovirus, EpsteinCBarr disease, varicella disease, human herpes simplex virus 6 and 7, parvovirus B19, respiratory syncytial trojan, hepatitis C trojan, and HIV. Particular antiviral therapies comprise val\/ganciclovir, acyclovir, foscarnet, high\dose intravenous immunoglobulins, and antiretroviral therapy administration in HIV illness.67 Of note, only a few are tested inside a trial placing for DCM, with limited randomized control data. In enterovirus\positive DCM individuals, spontaneous enterovirus elimination and elimination using interferon\ are both connected with clinical aswell as hemodynamic improvement.25 Of note, half of these patients spontaneously eliminated their enterovirus without specific treatment, suggesting that not merely the presence of viral genomes but also the replication status and viral load are important criteria for antiviral therapy.5 Retrospective research demonstrated that DCM patients with a high viral load might FRAX486 benefit from intravenous immunoglobulins.26 Although a randomized trial in 62 individuals with recent\onset DCM ( 6?weeks) didn’t show beneficial results on mortality and LVEF after 6 and 12?months, it lacked evaluation of viral infection and only 12% exhibited cardiac swelling.58 Currently, a single\center randomized controlled trial using high\dosage intravenous immunoglobulins (2.0?g/kg bodyweight) for chronic parvovirus B19Crelated DCM to lessen parvovirus B19 viral load was completed in August 2018, enrolling a total of 50 patients. (“type”:”clinical-trial”,”attrs”:”text message”:”NCT00892112″,”term_id”:”NCT00892112″NCT00892112). Toxic Triggers Toxic triggers are most known for functional and structural adjustments in the myocardium due to improved myocyte loss, which is irreversible largely.68, 69, 70 The main advice remains to reduce or withdraw the introduced toxic trigger such as excess alcohol, cocaine, or cancer therapy. Anthracyclines are most widely known because of their cardiotoxic impact. They express their cardiotoxicity via multiple systems: increase in oxidative stress, modulation in topoisomerase activity, alteration in the multidrug\resistant efflux proteins, and a decrease in mesenchymal progenitor cells.70 The decrease in mesenchymal progenitor cells decreases the cardioreparative capacity from the heart when it’s FRAX486 exposed to strain.71 A phase I randomized, placebo\controlled trial currently evaluates the safety and feasibility of administering mesenchymal progenitor cells to patients with anthracycline\induced DCM (“type”:”clinical-trial”,”attrs”:”text”:”NCT02509156″,”term_id”:”NCT02509156″NCT02509156). This trial will lay the foundation for a specific treatment relating to chemotherapy\induced DCM furthermore to fast treatment with general HF therapy.72 Multiple studies investigated the potential of prophylactic cardioprotective therapy using general HF medication or particular compounds such as dexrazoxane, an iron chelator that decreases the formation of superoxide radicals.73 However, current data do not support the routine prophylactic usage of HF treatment in sufferers receiving cardiotoxic chemotherapies. On the other hand, the usage of dexrazoxane is normally licensed for medical use in malignancy individuals undergoing anthracycline dosing more than 300?epirubicin or mg/m2 550?mg/m2. As a result, it might be considered in sufferers receiving these high dosages of cardiotoxic chemotherapy. 74 Ongoing research shall help better define the timing, dose, and length of prophylactic therapy to prevent HF onset in those patients in danger. Also, high\throughput screenings to recognize novel cardioprotective substances are ongoing, although non-e of them reached clinical trials yet.75 A cocaine use disorder can lead to a broad spectrum of cardiovascular complications.69 Careful diagnostics are therefore important when viewing an individual with suspicion of cocaine\related DCM. Cocaine exerts its toxic effect via multiple pathways: myocardial skin damage, impaired intracellular calcium mineral handling, apoptosis, boost of oxidative tension, and acute effects of the catecholamines. However, current treatment advice is to take care of based on the general HF suggestions, and trials looking into particular treatment regimens are lacking. Alcohol in low concentrations is beneficial for the heart, as opposed to the various other toxic sets off.68 Lengthy\term heavy alcohol consumption, however, can lead to alcoholic DCM, most likely via structural damage to the cardiomyocyte leading to apoptosis.68 Also, ethanol and its own metabolites are usually toxic for the mitochondria and sarcoplasm, altering calcium sensitivity on the myofilament level. Particular guidelines for the treatment of alcoholic DCM are lacking. Most studies focus on the clinical effect of alcohol decrease or comprehensive abstention.68 Patients who decreased their alcohol intake to moderate amounts had an identical survival and cardiac function recovery compared with complete alcohol abstainers.76 Overall, the alcoholic DCM individuals had a better survival weighed against the general band of DCM sufferers. Therefore, alcoholic beverages reduction in addition to general HF treatment is the keystone of therapy in alcoholic DCM. Electrical Triggers Arrhythmia\induced DCM is definitely a well\known potentially reversible condition where DCM is normally induced or mediated by atrial or ventricular arrhythmias and continues to be extensively examined previously.77 It may follow many types of cardiac arrhythmia: supraventricular tachyarrhythmias, ventricular tachycardia, or frequent ventricular ectopy. However, the medical diagnosis of arrhythmia\induced cardiomyopathy is normally frequently tough, as both are frequently diagnosed simultaneously. DCM in response to an arrhythmia may take weeks to years to build up, although rapid declines in ventricular function with development of HF symptoms are also observed in repeated (tachy)arrhythmias. If this helpful response continues to be absent, one must consider additional elements influencing the arrhythmic phenotype such as genetic mutations (ie, LMNA or SCN5A mutations).45 To date, the exact mechanisms of most genetic and nongenetic arrhythmias in DCM aren’t completely understood, but involve the loss of normal extracellular matrix and contractile dysfunction, alterations in cellular growth and viability, defects in Ca2+ handling, and neurohormonal activation results in DCM.77 Managing patients with suspected arrhythmic\induced DCM involves trying careful and aggressive control of rhythm and price, with the concentrate on arrhythmia elimination by catheter ablation whenever you can. Identifying the underlying condition is important to predict treatment response. In a study of 27 sufferers with regular PVCs (premature ventricular complexes) and DCM, 22 got improvement in LVEF pursuing PVC suppression; 5 didn’t. Four of the 5 sufferers with LVEF that didn’t improve had proof irreversible myocardial fibrosis discovered by past due gadolinium improvement CMR.78 A high PVC burden of 10?000?PVCs/d or 10% to 24% of total heartbeats/d can cause DCM. Reducing the PVC burden to 5000/d can improve LVEF.32 Moreover, removal of PVCs with ablation has been shown to improve LVEF, ventricular proportions, mitral regurgitation, and functional position. Within an observational series, ablation was more advanced than antiarrhythmic therapy in reducing PVCs and enhancing LVEF.79 In AF (atrial fibrillation), several tests evaluating rhythm control compared with rate control strategies have been conducted. Although they didn’t concentrate on arrhythmic\induced DCM, many lessons can be learned. In general, rhythm control will not provide a advantage in all\trigger mortality or worsening HF in comparison with price control, with related findings in stringent versus lenient rate control.35 In AF\mediated DCM, rhythm control is superior to rate control in enhancing LVEF, pro\brain natriuretic peptide levels, and standard of living.34 Moreover, in AF\mediated DCM, maintaining and restoring sinus rhythm can accelerate clinical recovery and reverse DCM over almost a year.33 Indeed, repair and maintenance of sinus rhythm by catheter ablation in individuals with congestive HF and AF significantly improves cardiac function, symptoms, exercise capacity, and quality of life.31 In atrial flutter, catheter ablation is recommended in arrhythmia\induced DCM, because price control is more challenging than in AF.36 Peripartum Cardiomyopathy Peripartum cardiomyopathy is a form of DCM with deterioration of cardiac function typically between the last trimester of pregnancy and up to 6?a few months postpartum. It really is recognized as a significant cause of being pregnant\related HF.80 To date, no evidence\based specific therapy is preferred in today’s guidelines. The exact disease pathophysiology is not known; however, high levels of cleaved 16\kDa N\terminal fragment of prolactin have already been been shown to be a significant mediator. Bromocriptine is certainly a dopamine\D2\receptor agonist and inhibits prolactin release. A pilot study in acute severe peripartum cardiomyopathy showed improvement in LVEF and prognosis when bromocriptine was added to standard HF therapy.29 A later on randomized multicenter clinical trial confirmed these findings and showed no factor between short\ and prolonged\term treatment with bromocriptine.30 Importantly, the trial didn’t include a non-use control group, leaving many unanswered queries. Ongoing observatory registries provides more info concerning adhere to\up and advantage of bromocriptine make use of. 81 Despite Western european encounter and recommendations, a placebo\controlled trial of bromocriptine for peripartum cardiomyopathy is very much indeed needed. Concentrating on Downstream Molecular Procedures Generating Disease Progression Despite many downstream functions driving the development to DCM overlap among different upstream causes, the severity and timing of these processes may vary. Medical result can be eventually determined by the interplay between trigger, host response, and therapeutic intervention. The alterations in cardiac rate of metabolism and fibrosis are well-known focuses on for treatment. In contrast, cardiomyocyte apoptosis, stretch, and harm are much less ideal for involvement because they’re often irreversible. However, cell therapy does provide potential as a future therapy to remedy cardiomyocyte reduction and harm. Cardiomyocyte Dysfunction DCM is seen as a biomechanical defects resulting in inadequate cardiac contraction. Positive inotropic brokers, such as dobutamine and milrinone, are indicated being a therapy in sufferers with end\stage HF and cardiogenic surprise.82 Milrinone is a bipyridine and inhibits phosphodiesterase\3, eventually resulting in an elevated cellular calcium mineral influx with subsequent activation of myocardial contractility. The OPTIME\CHF (Results of a Prospective Trial of Intravenous Milrinone for Exacerbations of Chronic Heart Failure) study was a big randomized trial to research the incremental worth of milrinone furthermore to standard HF therapy in individuals with acute HF.83 This scholarly research showed significant unwanted effects of milrinone, such as for example sustained hypotension and arrhythmias. A post hoc analysis demonstrated that milrinone was connected with higher mortality and rehospitalization in ischemic HF but was natural to helpful in nonischemic HF sufferers.84 However, we have to be careful in the interpretation of this post hoc analysis because the study was not initially designed for this purpose. For example, a subset analysis of the PRAISE (Propective Randomized Amlodipine Success Evaluation) I research showed a substantial decrease in mortality in the nonischemic subgroup treated with amlodipine.85 The well\designed PRAISE 2 research, whose purpose was to research the potential of amlodipine to reduce mortality in patients with nonischemic HF, failed to show this effect with trends in favor of placebo.86 There are no therapies that address the underlying biomechanical causes in DCM; among the newest therapies is aimed at raising the contractile function from the heart with minimal adverse effects on myocardial relaxation. The compound, named MYK\491, has been investigated inside a randomized presently, dual\blind, placebo\handled, crossover one\ascending dose stage 1b trial including DCM sufferers (“type”:”clinical-trial”,”attrs”:”text”:”NCT03447990″,”term_id”:”NCT03447990″NCT03447990). In contrast to milrinone, MYK\491 is an allosteric activator of myosin, therefore not increasing calcium levels in the cardiomyocytes and limiting potential side effects. If the tolerability and safety and echocardiographic actions of cardiac contractility are located to maintain positivity, MYK\491 will become pursued in potential research. A different inotropic compound is omecamtiv mecarbil, which demonstrated favorable hemodynamic leads to individuals with chronic HF inside a stage II medical trial.87 In individuals with acute HF, omecamtiv mecarbil did not meet the primary end point of dyspnea improvement.88 Both phase II trials (ATOMIC\AHF [Acute Treatment with Omecamtiv Mecarbil to Increase Contractility in Acute Heart Failure] and COSMIC\HF [Chronic Oral Research of Myosin activation to improve Contractility in Heart Failure]) demonstrated little increases in plasma troponin concentrations in treated individuals. Overall, there was no relationship with omecamtiv occurrence and concentrations of adverse events. The origin of the raised troponin concentrations is needs and unidentified to become addressed in bigger outcome trials. A phase III morbidity/mortality trial in patients with chronic HF (“type”:”clinical-trial”,”attrs”:”text”:”NCT02929329″,”term_id”:”NCT02929329″NCT02929329) is currently recruiting patients. Interestingly, omecamtiv mecarbil was demonstrated to augment cardiac contractility in DCM utilizing a DCM mouse model formulated with a tropomyosin mutation.89 This makes omecamtiv mecarbil a promising compound to take care of DCM patients with sarcomeric gene mutations. Cardiomyocyte Loss and Damage Stem cell therapy to pay for damaged/shed myocardial tissue goals to improve cardiac function in HF.39 Although the main focus of this approach was on ischemic HF, multiple clinical trials investigated the value of cell therapy in DCM, which is reviewed elsewhere extensively. 90 The many used way for delivery is intracoronary widely. Although shown to be safe and simple, these stem cells cannot reach the perfused myocardium inadequately. Also, homing and retention from the stem cells at the proper location continues to be difficult. A number of stem cell types are looked into in DCM: bone tissue marrowCderived, hematopoietic, and mesenchymal stem cells. In the beginning, cytokines and stem cells were investigated separately and both experienced shown beneficial effects on intermediate final result such as for example cardiac function.38, 91 REGENERATE\DCM was the initial stage II randomized, placebo\controlled trial teaching a substantial improvement in cardiac function, symptoms, and biochemical guidelines after a combined treatment of granulocyte colony\stimulating element and bone marrowCderived stem cell therapy.37 The full total outcomes from the REGENERATE\DCM can’t be translated to long\term outcome; therefore a stage III trial is essential to investigate these promising beneficial effects of combining granulocyte colony\revitalizing factor and bone marrowCderived stem cells in DCM. Modified Cardiac Metabolism One of the essential hallmarks in HF may be the decreased oxidative fat burning capacity from the cardiomyocyte.92 Therefore network marketing leads to a change from fatty acidity oxidation toward increased blood sugar rate of metabolism to fulfill the power demand from the contracting cardiac muscle tissue. A large part of the glucose utilization is used for anaerobic glycolysis, which is an energy\inefficient process. Eventually, the accumulating metabolic adjustments in HF will result in a power deficit, which maintains the development of the condition.92 Multiple substances have already been tested to intervene in different stages of metabolic changes, mainly because described inside a previous review extensively.92 One possible treatment system is to stimulate the center to use blood sugar as primary substrate by inhibiting the fatty acid oxidation. In this case, the heart will use its available oxygen in a far more effective method. Trimetazidine is usually a thiolase I inhibitor. Thiolase I catalyses the last step of the beta\oxidation in the mitochondria and its own inhibition will change the substrate usage toward glucose. Usage of trimetazidine furthermore to optimal treatment showed a substantial increase in LVEF in DCM.42 These findings were replicated in a later double\blinded study including DCM patients with diabetes mellitus.43 In addition, they showed the fact that inflammatory response was reduced also, as well as the physical performance of the patients was better. A recent meta\analysis of trimetazidine use in DCM showed its effectiveness relating to LVEF improvement and decrease in cardiac proportions.93 Although there is no long\term follow\up in DCM patients, a meta\evaluation showed TRIB3 a reduced risk for hospitalization but no influence on all\trigger mortality generally HF.94 Trimetazidine is the compound for metabolic support that is the most ready for larger\scale tests in DCM. Carnitine palmitoyl transferase is usually area of the acyl\carnitine shuttle from the mitochondria, which has a crucial function in fatty acidity transport. Etomoxir is definitely a compound that inhibits carnitine palmitoyl transferase and inhibits the beta\oxidation therefore, stimulating blood sugar as the principal substrate.92 The initial trial with etomoxir included 90% nonischemic DCM sufferers and demonstrated improvements in LVEF and clinical status.40 However, a larger follow\up study with a general HF population was terminated prematurely because of adverse side effects.95 Perhexiline is an alternative carnitine palmitoyl transferase inhibitor that demonstrated its value in hypertrophic cardiomyopathy and ischemic HF.96 Recently, a stage 2 randomized, twin\blinded, placebo\controlled research was published, only including nonischemic DCM sufferers.41 Although the study noted no benefit in cardiac function, the symptoms improved in individuals receiving perhexiline. More importantly, treatment with perhexiline increased the energy status of the heart without detectable changes of substrate utilization. This recommended that the procedure duration with this research was too brief or that the consequences of perhexiline are very much broader than just carnitine palmitoyl transferase inhibition. However, perhexiline was taken off the market in many countries because of its toxicity in patients with CYP2D6 polymorphisms, which constitutes a significant percentage in the white inhabitants. Although metabolic changes certainly are a main aftereffect of HF in the heart, therapy targeting these changes hasn’t seen clinical implementation yet, partly because of our incomplete knowledge regarding the specific metabolic changes in the heart. New strategies such as for example metabolomics will shed even more light for the molecular adjustments, which will help us to better identify the best sufferers and stage of the condition when metabolic support could possibly be the most effective. The existing available larger research used heterogeneous HF populations, leading to the blended outcomes probably. Preclinical and scientific data in the overall HF inhabitants with newer substances were encouraging,92 and investigating these newer compounds in DCM would be valuable. Cardiac Fibrosis Fibrosis is an essential element of tissues fix that follows tissues injury and is normally associated with inflammation. Intensifying fibrosis reflects a pathologic results and state in scarring and impairment of organ function. Myocardial fibrosis is normally a hallmark in end\stage DCM, you start with possibly reversible diffuse reactive fibrosis finally transitioning to irreversible focal scarring fibrosis.97 The main pathways and biological entities involved in the development of myocardial fibrosis will be the mineralocorticoid and transforming growth factor\ pathways, and nonstructural matrix micro\RNAs and protein.98 To date, the influence of gene mutations and extra environmentally obtained factors within the extent of myocardial fibrosis is largely unknown. Some insights have been gained by analyzing subsets of individuals with DCM such as people that have mutations where early skin damage fibrosis is connected with increased threat of arrhythmias.45 In this respect, focusing on fibrosis could be split into 2 types. The first category is preventing or delaying the onset of myocardial fibrosis, with early detection of potentially treatable causes to avoid further progression toward irreversible fibrosis in DCM patients or those vulnerable to developing DCM. The next category can be regression or stabilization of established myocardial fibrosis through targeting of key molecular processes. Although these key molecular processes can be targeted in several ways, many strategies usually do not focus on fibrosis specifically. Mineralocorticoid receptor antagonists, already section of regular HF therapy, may be interesting antifibrotic drugs because they work about extracellular matrix remodeling, lower collagen biosynthesis biomarkers, and improve results in individuals with HF and DCM.44, 99 In DCM patients, myocardial fibrosis on a histologic level can be reduced using mineralocorticoid receptor antagonists accompanied by a decrease in collagen biosynthesis biomarkers.44 Moreover, myocardial gal\3 (galectin\3) plasma concentrations are increased by aldosterone and angiotensin II early in the fibrotic cascade and may be decreased by mineralocorticoid receptor antagonists, in keeping with an impact of these real estate agents on cells pathology.99 Oher potential therapeutic targets include transforming growth factor\, interleukins, and wnt signaling, because they can transform quiescent fibroblasts into active collagen\producing myofibroblasts.100 Although the molecular transforming growth factor\ pathway is important in practically all types of fibrosis, its pleiotropic results produce it an unattractive target. Cardiotrophin\1, an associate from the IL\6 superfamily, is usually a profibrotic factor and is increased in the myocardium of sufferers with HF of different causes, including DCM.101 Finally, inhibition of wnt signaling reduced fibrosis and cardiac recovery in mice.102 Up to now, pharmacological research are performed only in gal\3 and wnt signaling. Gal\3 inhibitors consist of carbohydrates such as for example customized citrus pectin, which is being used in an ongoing trial including patients with high blood pressure to lessen cardiac fibrosis (“type”:”clinical-trial”,”attrs”:”text message”:”NCT01960946″,”term_id”:”NCT01960946″NCT01960946). Oddly enough, a stage 1 trial utilizing a monoclonal antibody called vantictumab, which inhibits downstream signaling of wnt, was completed in December 2017, results which are still getting anticipated (“type”:”clinical-trial”,”attrs”:”text message”:”NCT01973309″,”term_id”:”NCT01973309″NCT01973309). Hence, focusing on the angiotensin II\galectin\3\IL\6 axis using mineralocorticoid receptor antagonists or Gal\3 inhibitors or inhibiting wnt signaling pathway by vantictumab could specifically reduce cardiac fibrosis. Matricellular proteins have a potential in antifibrotic therapies, because they can degrade collagen and modulate the function of cardiac fibroblasts, inflammatory cells, and cardiomyocytes. Recently, the first proof of principle for the therapeutic potential provides advanced for osteoglycin. Lack of osteoglycin in mice decreased collagen combination\linking in the infarct scar after myocardial infarction, therefore increasing cardiac rupture and dilatation. FRAX486 Osteoglycin administration through gene therapy improved collagen maturation and prevented LV dilatation and dysfunction after myocardial infarction thereby.103 To date, no individual trials exist. Various little noncoding RNAs (miRs), posttranscriptional regulators of gene expression, get excited about cardiac fibrosis. Myocardial fibrosis in sufferers with varied cardiac disease is definitely accompanied by improved manifestation of profibrotic miRs including miR\21, miR\208a, and miR\499\5p, and decreased manifestation of antifibrotic miRs such as for example miR\29, miR 19b, miR\1, miR\133a, and miR\122.98 Moreover, miRNAs could be easily manipulated and offer a book class of antifibrotic agents. Although no human being tests including HF sufferers getting miRs are executed however, the antifibrotic miR\122 reached stage II trial for dealing with hepatitis.104 Thus, the advancement of miR\based therapeutics in to the clinical testing of HF is coming. Besides quantity may be the quality of myocardial fibrosis, with regards to collagen mix\linking, which can be an equally important feature. 97 Excessively mix\linked collagen is challenging to degrade and affects extracellular matrix turnover critically. Syndecan\4\osteopontin\Lysyl Oxidase\Like axis is important in the formation of insoluble cross\linked collagen, and therapeutic strategies that focus on such pathways could ameliorate the consequences of myocardial fibrosis also.105 A monoclonal antibody against Lysyl Oxidase\Like 2, called simtuzumab, is currently being tested in a phase IIb trial in subjects with compensated cirrhosis secondary to nonalcoholic steatohepatitis (“type”:”clinical-trial”,”attrs”:”text”:”NCT01672879″,”term_id”:”NCT01672879″NCT01672879). So far, no clinical trials in cardiac illnesses have already been initiated. Interestingly, in neuro-scientific idiopathic pulmonary fibrosis, 2 antifibrotic medicines, pirfenidone and nintedanib, have been been shown to be safe and effective in the treatment of idiopathic pulmonary fibrosis and are currently recommended for use in these patients.106 Nintedanib is a tyrosine kinase inhibitor that targets growth factor pathways and pirfenidone has a amount of anti\inflammatory and antifibrotic results, including inhibition of collagen synthesis, downregulation of transforming growth factor\ and tumor necrosis factor\, and a decrease in fibroblast proliferation.107 Currently, a stage II trial has been conducted to evaluate the efficacy and safety of pirfenidone in patients with HF and preserved ejection fraction (“type”:”clinical-trial”,”attrs”:”text”:”NCT02932566″,”term_id”:”NCT02932566″NCT02932566). Clinical Implications and Future Perspectives The greatest pitfall of evidence\based cause\directed therapy is the few patients contained in studies ( 100) (Table?2). That is mainly because from the heterogeneous character of DCM and incomplete characterization of cause, which limits the real amounts of particular subgroups of patients. Often echocardiographic evaluation is available without CMR, and endomyocardial biopsy is not standard in all countries/practices. Although the quality of most studies is good, almost all remain one\center experience. As a result, scientific implications are limited. The just guideline\changing therapies over the past few years were tested in general HF, of which DCM is usually a little subset. This retains the current dogma of nonpersonalized treatment. Understanding the molecular consequences of genetic DCM will provide novel and specific treatment targets. DCM is an excellent example, as the changed intracellular signaling is normally well studied. As a result, multiple therapies can hinder the disease training course, upstream and downstream (Number?3). With the high prevalence of em TTN /em \connected DCM, its molecular and phenotypical signature is becoming even more apparent. This will also provide restorative options for these specific patients. Although we have a broad collection of therapies, we’ve to good\tune the treatment based on upstream triggers and their downstream active processes (Figure?3). Open in a separate window Figure 3 Two types of genetic mutations resulting in dilated cardiomyopathy and their potential treatment focuses on interfering with disease development at different amounts. Hereditary diagnostics adds a different dimension to the spectrum. The upsurge in genotypeCphenotype knowledge will benefit prevention and a personalized approach greatly. Early detection and preventive medicine will be a significant focus of upcoming research. Clinical and hereditary screening of DCM relatives is a perfect example for identifying carriers of a pathogenic mutation who are at risk for developing the phenotype. Herein, global longitudinal stress may provide a good device to detect subclinical disease in healthful DCM family members and moreover carries prognostic relevance. However, there is great clinical variability and incomplete penetrance within families because of elements we don’t realize yet. Moreover, whether prophylactic treatment is able and beneficial to prevent disease onset is usually unidentified. This is a significant area of analysis since it will significantly benefit patient care and provide reassurance among genetic DCM families. Another important clinical issue is the security of medication withdrawal in currently asymptomatic DCM sufferers with complete cardiac recovery. Notably, DCM individuals are relatively hesitant and youthful to consider lifelong medicine without continued beneficial results. A recent open up\label, randomized, pilot trial suggested that 40% of individuals with recovered DCM will have a relapse within 6?a few months after phased medicine discontinuation.108 However, this also showed that 60% of DCM sufferers did not have got a relapse over that time period. Future study should focus on identifying subgroups of DCM sufferers in whom pharmacological treatment for HF could be properly withdrawn. One particular feasible marker for determining subgroups can be global longitudinal stress evaluation on echocardiography. A recently available study showed that 79% of recovered HF patients still had an abnormal global longitudinal strain, despite normalization of geometry and/or LVEF.109 Furthermore, the abnormal global longitudinal strain is a predictor for worse clinical outcome generally recovered HF patients. The best goal is to link the main element disease\traveling mechanisms in DCM patients with specific therapies. These targeted therapies will become most beneficial in those patients who do not improve sufficiently using the established guideline\directed medical therapies.17 This will collection the stage for even more in\depth pheno\ and genotyping using the proposed diagnostic DCM workup (Figure?2). Therefore, standardized classification of DCM subclasses according to the upstream cause and downstream molecular processes at the time of clinical evaluation is crucial. Herein, the Morphology, Organ Involvement, Hereditary, Etiology, Stage of disease classification can offer the first guidelines toward standardized classification of the subclasses who might advantage most from targeted therapy (eg, immunosuppressive, antifibrotic therapies). Once global standardized classification and following stratification validates the suggested up\ and downstream subclasses, multicenter trials using targeted therapies adjunctive to established guideline\directed medical therapy can be initiated to show their incremental worth. In the foreseeable future, recruitment should begin in global professional centers using the availability of advanced imaging systems (eg, CMR), genetic evaluation, and preferably myocardial biopsy. Treatment efficacy should be monitored using founded (natriuretic peptides, troponin) and relevant circulating biomarkers for the precise therapy (eg, inflammatory markers for immunosuppression, fibrosis markers for antifibrotic therapy). Furthermore, assessment of brief\term (6C12?a few months) functional improvement such as for example HF questionnaires, workout tolerance, and cardiac function together with hard end points including HF hospitalization, existence\threatening arrhythmias, implantation of ventricular aid device, center transplantation, and loss of life are crucial to judge long\term beneficial ramifications of the targeted therapy. Conclusions DCM takes its large clinical phenotype that arises from a final common response to a number of genetic and environmentally acquired insults. Detecting the underlying dominating cause and the ongoing downstream pathophysiological processes is the key to initiate targeted treatment strategies. Several small targeted tests have been carried out, but as of the era has arrived to conduct large now, multicenter tests that bring about breakthroughs in targeted treatment that boosts prognosis in these individuals. Sources of Funding This work was supported by the European Union Commission Seventh Framework Programme under grant agreement No. 305507 (HOMAGE). Kootstra Talent Fellowship of the Maastricht UMC+ to Dr Hazebroek. Dr Ware received funding from Wellcome Trust (107469/Z/15/Z). Dr Prasad offers received financing from British Center Basis, Rosetrees, and Alexander Jansons Basis. We also acknowledge the support from holland Cardiovascular Research Initiative, an effort with support from the Dutch Center Foundation, CVON2011\Area, CVON2016\Early HFPEF, and CVON 2017\ShePREDICTS. This analysis is cofinanced being a PPP Allowance Analysis and Innovation by the Ministry of Economic Affairs within Top Sector Life Sciences & Health. Disclosures Dr Ware did consultancy work for MyoKardia. The remaining authors haven’t any disclosures to record. Notes J Am Center Assoc. 2019;8:e012514 DOI: 10.1161/JAHA.119.012514. [PMC free of charge content] [PubMed] [CrossRef] [Google Scholar]. reducing publicity; use of cardioprotective brokers in anthracycline toxicity (dexrazoxane)InflammationEMB, blood sampling (eg, sIL2\R, CRP, galectin\3), CMRImmune cell infiltration in EMB, increased T2\signal, raised CRP/ESRPro\inflammatory pathways (eg IL\1)Autoimmune diseaseEMB, blood sampling, imaging, autoantibody display screen, existence of extracardiac featuresPresence of irritation and positive autoantibody titersAuto\antibodies (eg, (1)\AABs)ViralEMBCardiotropic pathogen presence using a viral weight 500?copies/g DNAVirus and subsequent cardiac inflammationElectrical12\lead ECG, ambulatory ECG monitoring 10?000 to 25?000?PVCs/d; (supra)ventricular tachycardiaAbnormal electrical pathwaysPeripartum(history of) PregnancyDisease onset during pregnancy up to 6?mo postpartumCleaved 16?kDa N\terminal fragment of prolactinCardiac metabolismBlood, tissue and/or urine metabolomicsElevated acylcarnitines, upsurge in ketone bodiesMultiple strategies possible interfering using the metabolic substrate change (mainly involving mitochondrial pathways)Cardiac fibrosisEMB, CMR, bloodIncreased CFV; midmyocardial LE; elevated fibrosis bloodstream markersRAAS\pathway; angiotensin II\galectin\3\interleukin\6 axis; matricellular protein; syndecan\4\osteopontin\lysyl oxidase\like axis Open in a separate window AAB shows autoantibodies; CFV, collagen portion volume; CMR, cardiac magnetic resonance; CRP, C\reactive protein; DCM, dilated cardiomyopathy; EMB, endomyocardial biopsy; ESR, erythrocyte sedimentation rate; ICD, implantable cardiac defibrillator; LE, past due enhancement; PVC, early ventricular complicated; RAAS, renin\angiotensin\aldosterone program; WES, entire exome sequencing. Open in a separate window Number 2 Total diagnostic workup of a dilated cardiomyopathy patient to characterize the cardiac function and underlying cause. CRP signifies C\reactive proteins; sIL2\R, soluble interleukin 2 receptor. PRESENT STATE of Guide\Powered Therapies The existing treatment of DCM sufferers does not vary from general HF management, with the pharmacological cornerstone consisting of \blockers, RAS (renin\angiotensin system) inhibitors, aldosterone antagonists, and diuretics.17 Probably one of the most recent guide\changing breakthroughs generally HF treatment was the introduction from the angiotensin receptor\neprilysin inhibitor sacubitril\valsartan.18 The major PARADIGM (Prospective Comparison of ARNI with ACEI to Determine Effect on Global Mortality and Morbidity in Heart Failure) trial showed a risk reduced amount of loss of life and HF hospitalization in chronic HF individuals treated with sacubitril\valsartan instead of enalapril. About 60% of the included individuals experienced an ischemic cause, making the drug still relatively brand-new in neuro-scientific DCM. The outcomes from the PARADIGM trial have already been examined post hoc in a number of subgroups.19 A chance remains to execute yet another post hoc analysis in mere nonischemic HF patients to judge its effect with this subgroup. Targeting the Underlying Cause of DCM: Identifying Upstream Causes In DCM it would be the goal to initiate therapies targeting essential molecular processes powered by upstream causes. Understanding regarding factors behind DCM has extended significantly within the last few years. The increasing number of patients in registries and combining international databases provides us with valuable information regarding clinical demonstration and prognosis of particular causes. Current recommendations do not consist of treatment strategies fond of key molecular procedures driven by upstream causes, because evidence is mostly coming from single\center pilot and retrospective cohort studies (Desk?2). This is the time to help make the next thing toward multicenter, randomized tests (Desk?3). Desk 2 Overview of All Studies Investigating Cause\Directed Treatments in Dilated Cardiomyopathy Patients DCM patients Unravel molecular outcomes of particular gene mutations Gene modification therapies towards a scientific application InflammationNo guide\directed therapy; although there is usually evidence from retrospective studies showing benefit from immunosuppressionPhase 2B RCT in acute myocarditis using anakinra vs regular treatment Multicenter RCT using immunosuppressive therapy in inflammatory DCM Car\immunityNo guide\aimed therapy; although there is certainly evidence showing reap the benefits of immunoadsorption Multicenter RCT using immunoadsorption in DCM with cardiotoxic autoantibodies ViralNo guideline\directed therapy; although there are retrospective studies and case reports showing benefit from IVIg Phase 3 RCT using IVIg for chronic PVB19\related DCM RCT using particular antiviral therapies (val/\ganciclovir) Multicenter RCT for IVIg if stage 3 trial is certainly positive ToxicNo guide\aimed therapy; withholding or reducing publicity has been proven to be.