S-glutathionylation of cryptic cysteines enhances titin elasticity by blocking protein folding.

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric-oxide-signaling cascade that increases cardiac muscle elasticity. However, it is unknown how S-glutathionylation may regulate the elasticity of titin and cardiac tissue. Here, we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore, we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechanochemical modulation of the elasticity of human cardiomyocytes. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.

Small conductance calcium-activated potassium channel contributes to stress induced endothelial dysfunctions.

Patients with Takotsubo syndrome displayed endothelial dysfunction, but underlying mechanisms have not been fully clarified. This study aimed to explore molecular signalling responsible for catecholamine excess induced endothelial dysfunction. Human cardiac microvascular endothelial cells were challenged by epinephrine to mimic catecholamine excess. Patch clamp, FACS, ELISA, PCR, and immunostaining were employed for the study. Epinephrine (Epi) enhanced small conductance calcium-activated potassium channel current (ISK1-3) through activating α1 adrenoceptor. Phenylephrine enhanced edothelin-1 (ET-1) and reactive oxygen species (ROS) production, and the effects involved contribution of ISK1-3. H2O2 enhanced ISK1-3 and ET-1 production. Enhancing ISK1-3 caused a hyperpolarization, which increases ROS and ET-1 production. BAPTA partially reduced phenylephrine-induced enhancement of ET-1 and ROS, suggesting that α1 receptor activation can enhance ROS/ET-1 generation in both calcium-dependent and calcium-independent ways. The study demonstrates that high concentration catecholamine can activate SK1-3 channels through α1 receptor-ROS signalling and increase ET-1 production, facilitating vasoconstriction.

Effects of Atrial Fibrillation on the Human Ventricle.

RATIONALE: Atrial fibrillation (AF) and heart failure often coexist, but their interaction is poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to left ventricular (LV) dysfunction. OBJECTIVE: This study investigates the effects and molecular mechanisms of AF on the human LV. METHODS AND RESULTS: Ventricular myocardium from patients with aortic stenosis and preserved LV function with sinus rhythm or rate-controlled AF was studied. LV myocardium from patients with sinus rhythm and patients with AF showed no differences in fibrosis. In functional studies, systolic Ca2+ transient amplitude of LV cardiomyocytes was reduced in patients with AF, while diastolic Ca2+ levels and Ca2+ transient kinetics were not statistically different. These results were confirmed in LV cardiomyocytes from nonfailing donors with sinus rhythm or AF. Moreover, normofrequent AF was simulated in vitro using arrhythmic or rhythmic pacing (both at 60 bpm). After 24 hours of AF-simulation, human LV cardiomyocytes from nonfailing donors showed an impaired Ca2+ transient amplitude. For a standardized investigation of AF-simulation, human iPSC-cardiomyocytes were tested. Seven days of AF-simulation caused reduced systolic Ca2+ transient amplitude and sarcoplasmic reticulum Ca2+ load likely because of an increased diastolic sarcoplasmic reticulum Ca2+ leak. Moreover, cytosolic Na+ concentration was elevated and action potential duration was prolonged after AF-simulation. We detected an increased late Na+ current as a potential trigger for the detrimentally altered Ca2+/Na+-interplay. Mechanistically, reactive oxygen species were higher in the LV of patients with AF. CaMKII (Ca2+/calmodulin-dependent protein kinase IIδc) was found to be more oxidized at Met281/282 in the LV of patients with AF leading to an increased CaMKII activity and consequent increased RyR2 phosphorylation. CaMKII inhibition and ROS scavenging ameliorated impaired systolic Ca2+ handling after AF-simulation. CONCLUSIONS: AF causes distinct functional and molecular remodeling of the human LV. This translational study provides the first mechanistic characterization and the potential negative impact of AF in the absence of tachycardia on the human ventricle.

Protein Kinase D Plays a Crucial Role in Maintaining Cardiac Homeostasis by Regulating Post-Translational Modifications of Myofilament Proteins.

Protein kinase D (PKD) enzymes play important roles in regulating myocardial contraction, hypertrophy, and remodeling. One of the proteins phosphorylated by PKD is titin, which is involved in myofilament function. In this study, we aimed to investigate the role of PKD in cardiomyocyte function under conditions of oxidative stress. To do this, we used mice with a cardiomyocyte-specific knock-out of Prkd1, which encodes PKD1 (Prkd1loxP/loxP; αMHC-Cre; PKD1 cKO), as well as wild type littermate controls (Prkd1loxP/loxP; WT). We isolated permeabilized cardiomyocytes from PKD1 cKO mice and found that they exhibited increased passive stiffness (Fpassive), which was associated with increased oxidation of titin, but showed no change in titin ubiquitination. Additionally, the PKD1 cKO mice showed increased myofilament calcium (Ca2+) sensitivity (pCa50) and reduced maximum Ca2+-activated tension. These changes were accompanied by increased oxidation and reduced phosphorylation of the small myofilament protein cardiac myosin binding protein C (cMyBPC), as well as altered phosphorylation levels at different phosphosites in troponin I (TnI). The increased Fpassive and pCa50, and the reduced maximum Ca2+-activated tension were reversed when we treated the isolated permeabilized cardiomyocytes with reduced glutathione (GSH). This indicated that myofilament protein oxidation contributes to cardiomyocyte dysfunction. Furthermore, the PKD1 cKO mice exhibited increased oxidative stress and increased expression of pro-inflammatory markers interleukin (IL)-6, IL-18, and tumor necrosis factor alpha (TNF-α). Both oxidative stress and inflammation contributed to an increase in microtubule-associated protein 1 light chain 3 (LC3)-II levels and heat shock response by inhibiting the mammalian target of rapamycin (mTOR) in the PKD1 cKO mouse myocytes. These findings revealed a previously unknown role for PKD1 in regulating diastolic passive properties, myofilament Ca2+ sensitivity, and maximum Ca2+-activated tension under conditions of oxidative stress. Finally, we emphasized the importance of PKD1 in maintaining the balance of oxidative stress and inflammation in the context of autophagy, as well as cardiomyocyte function.

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx).

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

Cardiac mechanisms of the beneficial effects of SGLT2 inhibitors in heart failure: Evidence for potential off-target effects.

Sodium glucose cotransporter 2 inhibitors (SGLT2i) constitute a promising drug treatment for heart failure patients with either preserved or reduced ejection fraction. Whereas SGLT2i were originally developed to target SGLT2 in the kidney to facilitate glucosuria in diabetic patients, it is becoming increasingly clear that these drugs also have important effects outside of the kidney. In this review we summarize the literature on cardiac effects of SGLT2i, focussing on pro-inflammatory and oxidative stress processes, ion transport mechanisms controlling sodium and calcium homeostasis and metabolic/mitochondrial pathways. These mechanisms are particularly important as disturbances in these pathways result in endothelial dysfunction, diastolic dysfunction, cardiac stiffness, and cardiac arrhythmias that together contribute to heart failure. We review the findings that support the concept that SGLT2i directly and beneficially interfere with inflammation, oxidative stress, ionic homeostasis, and metabolism within the cardiac cell. However, given the very low levels of SGLT2 in cardiac cells, the evidence suggests that SGLT2-independent effects of this class of drugs likely occurs via off-target effects in the myocardium. Thus, while there is still much to be understood about the various factors which determine how SGLT2i affect cardiac cells, much of the research clearly demonstrates that direct cardiac effects of these SGLT2i exist, albeit mediated via SGLT2-independent pathways, and these pathways may play a role in explaining the beneficial effects of SGLT2 inhibitors in heart failure.

Leveraging clinical epigenetics in heart failure with preserved ejection fraction: a call for individualized therapies.

Described as the 'single largest unmet need in cardiovascular medicine', heart failure with preserved ejection fraction (HFpEF) remains an untreatable disease currently representing 65% of new heart failure diagnoses. HFpEF is more frequent among women and associates with a poor prognosis and unsustainable healthcare costs. Moreover, the variability in HFpEF phenotypes amplifies complexity and difficulties in the approach. In this perspective, unveiling novel molecular targets is imperative. Epigenetic modifications-defined as changes of DNA, histones, and non-coding RNAs (ncRNAs)-represent a molecular framework through which the environment modulates gene expression. Epigenetic signals acquired over the lifetime lead to chromatin remodelling and affect transcriptional programmes underlying oxidative stress, inflammation, dysmetabolism, and maladaptive left ventricular remodelling, all conditions predisposing to HFpEF. The strong involvement of epigenetic signalling in this setting makes the epigenetic information relevant for diagnostic and therapeutic purposes in patients with HFpEF. The recent advances in high-throughput sequencing, computational epigenetics, and machine learning have enabled the identification of reliable epigenetic biomarkers in cardiovascular patients. Contrary to genetic tools, epigenetic biomarkers mirror the contribution of environmental cues and lifestyle changes and their reversible nature offers a promising opportunity to monitor disease states. The growing understanding of chromatin and ncRNAs biology has led to the development of several Food and Drug Administration approved 'epidrugs' (chromatin modifiers, mimics, anti-miRs) able to prevent transcriptional alterations underpinning left ventricular remodelling and HFpEF. In the present review, we discuss the importance of clinical epigenetics as a new tool to be employed for a personalized management of HFpEF.

Leveraging clinical epigenetics in heart failure with preserved ejection fraction: a call for individualized therapies.

Described as the 'single largest unmet need in cardiovascular medicine', heart failure with preserved ejection fraction (HFpEF) remains an untreatable disease currently representing 65% of new heart failure diagnoses. HFpEF is more frequent among women and associates with a poor prognosis and unsustainable healthcare costs. Moreover, the variability in HFpEF phenotypes amplifies complexity and difficulties in the approach. In this perspective, unveiling novel molecular targets is imperative. Epigenetic modifications-defined as changes of DNA, histones, and non-coding RNAs (ncRNAs)-represent a molecular framework through which the environment modulates gene expression. Epigenetic signals acquired over the lifetime lead to chromatin remodelling and affect transcriptional programmes underlying oxidative stress, inflammation, dysmetabolism, and maladaptive left ventricular remodelling, all conditions predisposing to HFpEF. The strong involvement of epigenetic signalling in this setting makes the epigenetic information relevant for diagnostic and therapeutic purposes in patients with HFpEF. The recent advances in high-throughput sequencing, computational epigenetics, and machine learning have enabled the identification of reliable epigenetic biomarkers in cardiovascular patients. Contrary to genetic tools, epigenetic biomarkers mirror the contribution of environmental cues and lifestyle changes and their reversible nature offers a promising opportunity to monitor disease states. The growing understanding of chromatin and ncRNAs biology has led to the development of several Food and Drug Administration approved 'epidrugs' (chromatin modifiers, mimics, anti-miRs) able to prevent transcriptional alterations underpinning left ventricular remodelling and HFpEF. In the present review, we discuss the importance of clinical epigenetics as a new tool to be employed for a personalized management of HFpEF.

Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G).

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

Linagliptin prevents left ventricular stiffening by reducing titin cleavage and hypophosphorylation.

The metabolic syndrome (MetS) is an escalating problem worldwide, causing left ventricular stiffening, an early characteristic of diastolic dysfunction for which no treatment exists. As diastolic dysfunction and stiffening in MetS patients are associated with increased circulating dipeptidyl peptidase-4 (DPP-4) levels, we investigated whether the clinically approved DPP-4 inhibitor linagliptin reduces left ventricular stiffness in MetS-induced cardiac disease. Sixteen-week-old obese ZSF1 rats, displaying the MetS and left ventricular stiffness, received linagliptin-supplemented or placebo diet for four weeks. Linagliptin significantly reduced obesity, hyperlipidaemia, and hyperglycaemia and improved left ventricular relaxation. This improved relaxation was related to decreased cardiac fibrosis and cardiomyocyte passive stiffness (Fpassive ). The reduced Fpassive was the result of titin isoform switching from the stiff N2B to the more flexible N2BA and increased phosphorylation of total titin and specifically its N2Bus region (S4080 and S3391). Importantly, DPP-4 directly cleaved titin in vitro, resulting in an increased Fpassive , which was prevented by simultaneous administration of linagliptin. In conclusion, linagliptin improves left ventricular stiffness in obese ZSF1 rats by preventing direct DPP4-mediated titin cleavage, as well as by modulating both titin isoform levels and phosphorylation. Reducing left ventricular stiffness by administering linagliptin might prevent MetS-induced early diastolic dysfunction in human.

Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease.

Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

SGLT2 Inhibitors and Their Mode of Action in Heart Failure-Has the Mystery Been Unravelled?

PURPOSE OF REVIEW: SGLT2 inhibitors (SGLT2i) are new drugs for patients with heart failure (HF) irrespective of diabetes. However, the mechanisms of SGLT2i in HF remain elusive. This article discusses the current clinical evidence for using SGLT2i in different types of heart failure and provides an overview about the possible underlying mechanisms. RECENT FINDINGS: Clinical and basic data strongly support and extend the use of SGLT2i in HF. Improvement of conventional secondary risk factors is unlikely to explain the prognostic benefits of these drugs in HF. However, different multidirectional mechanisms of SGLT2i could improve HF status including volume regulation, cardiorenal mechanisms, metabolic effects, improved cardiac remodelling, direct effects on cardiac contractility and ion-homeostasis, reduction of inflammation and oxidative stress as well as an impact on autophagy and adipokines. Further translational studies are needed to determine the mechanisms of SGLT2i in HF. However, basic and clinical evidence encourage the use of SGLT2i in HFrEF and possibly HFpEF.

Cardiomyocyte Dysfunction in Inherited Cardiomyopathies.

Inherited cardiomyopathies form a heterogenous group of disorders that affect the structure and function of the heart. Defects in the genes encoding sarcomeric proteins are associated with various perturbations that induce contractile dysfunction and promote disease development. In this review we aimed to outline the functional consequences of the major inherited cardiomyopathies in terms of myocardial contraction and kinetics, and to highlight the structural and functional alterations in some sarcomeric variants that have been demonstrated to be involved in the pathogenesis of the inherited cardiomyopathies. A particular focus was made on mutation-induced alterations in cardiomyocyte mechanics. Since no disease-specific treatments for familial cardiomyopathies exist, several novel agents have been developed to modulate sarcomere contractility. Understanding the molecular basis of the disease opens new avenues for the development of new therapies. Furthermore, the earlier the awareness of the genetic defect, the better the clinical prognostication would be for patients and the better the prevention of development of the disease.

De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents.

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient's myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

CaMKII activity contributes to homeometric autoregulation of the heart: A novel mechanism for the Anrep effect.

KEY POINTS: The Anrep effect represents the alteration of left ventricular (LV) contractility to acutely enhanced afterload in a few seconds, thereby preserving stroke volume (SV) at constant preload. As a result of the missing preload stretch in our model, the Anrep effect differs from the slow force response and has a different mechanism. The Anrep effect demonstrated two different phases. First, the sudden increased afterload was momentary equilibrated by the enhanced LV contractility as a result of higher power strokes of strongly-bound myosin cross-bridges. Second, the slightly delayed recovery of SV is perhaps dependent on Ca2+ /calmodulin-dependent protein kinase II activation caused by oxidation and myofilament phosphorylation (cardiac myosin-binding protein-C, myosin light chain 2), maximizing the recruitment of available strongly-bound myosin cross-bridges. Short-lived oxidative stress might present a new facet of subcellular signalling with respect to cardiovascular regulation. Relevance for human physiology was demonstrated by echocardiography disclosing the Anrep effect in humans during handgrip exercise. ABSTRACT: The present study investigated whether oxidative stress and Ca2+ /calmodulin-dependent protein kinase II (CaMKII) activity are involved in triggering the Anrep effect. LV pressure-volume (PV) analyses of isolated, preload controlled working hearts were performed at two afterload levels (60 and 100 mmHg) in C57BL/6N wild-type (WT) and CaMKII-double knockout mice (DKOCaMKII ). In snap-frozen WT hearts, force-pCa relationship, H2 O2 generation, CaMKII oxidation and phosphorylation of myofilament and Ca2+ handling proteins were assessed. Acutely raised afterload showed significantly increased wall stress, H2 O2 generation and LV contractility in the PV diagram with an initial decrease and recovery of stroke volume, whereas end-diastolic pressure and volume, as well as heart rate, remained constant. Afterload induced increase in LV contractility was blunted in DKOCaMKII -hearts. Force development of single WT cardiomyocytes was greater with elevated afterload at submaximal Ca2+ concentration and associated with increases in CaMKII oxidation and phosphorylation of cardiac-myosin binding protein-C, myosin light chain and Ca2+ handling proteins. CaMKII activity is involved in the regulation of the Anrep effect and associates with stimulation of oxidative stress, presumably starting a cascade of CaMKII oxidation with downstream phosphorylation of myofilament and Ca2+ handling proteins. These mechanisms improve LV inotropy and preserve stroke volume within few seconds.

Prophylactic, single-drug cardioprotection in a comparative, experimental study of doxorubicin-induced cardiomyopathy.

BACKGROUND: Cardiomyopathy is a common side effect of doxorubicin (DOX) chemotherapy. Despite intensive research efforts in the field, there is still no evidence available for routine cardioprotective prophylaxis to prevent cardiotoxicity in the majority of oncological patients at low risk of cardiovascular disease. We have recently demonstrated the advantages of a prophylactic, combined heart failure therapy in an experimental model of DOX-induced cardiomyopathy. In the current work, we focus on individually applied prophylactic medications studied in the same translational environment to clarify their distinct roles in the prevention of DOX cardiotoxicity. METHODS: Twelve-week-old male Wistar rats were divided into 5 subgroups. Prophylactic ß-blocker (BB, bisoprolol), angiotensin-converting enzyme inhibitor (ACEI, perindopril) or aldosterone antagonist (AA, eplerenone) treatments were applied 1 week before DOX administration, then 6 cycles of intravenous DOX chemotherapy were administered. Rats receiving only intravenous DOX or saline served as positive and negative controls. Blood pressure, heart rate, body weight, and echocardiographic parameters were monitored in vivo. Two months after the last DOX administration, the animals were sacrificed, and their heart and serum samples were frozen in liquid nitrogen for histological, mechanical, and biochemical measurements. RESULTS: All prophylactic treatments increased the survival of DOX-receiving animals. The lowest mortality rates were seen in the BB and ACEI groups. The left ventricular ejection fraction was only preserved in the BB group. The DOX-induced increase in the isovolumetric relaxation time could not be prevented by any prophylactic treatment. A decreased number of apoptotic nuclei and a preserved myocardial ultrastructure were found in all groups receiving prophylactic cardioprotection, while the DOX-induced fibrotic remodelling and the increase in caspase-3 levels could only be substantially prevented by the BB and ACEI treatments. CONCLUSION: Primary prophylaxis with cardioprotective agents like BB or ACEI has a key role in the prevention of DOX-induced cardiotoxicity in healthy rats. Future human studies are necessary to implement this finding in the clinical management of oncological patients free of cardiovascular risk factors.

Diabetes-Induced Cardiomyocyte Passive Stiffening Is Caused by Impaired Insulin-Dependent Titin Modification and Can Be Modulated by Neuregulin-1.

RATIONALE: Increased titin-dependent cardiomyocyte tension is a hallmark of heart failure with preserved ejection fraction associated with type-2 diabetes mellitus. However, the insulin-related signaling pathways that modify titin-based cardiomyocyte tension, thereby contributing to modulation of diastolic function, are largely unknown. OBJECTIVE: We aimed to determine how impaired insulin signaling affects titin expression and phosphorylation and thus increases passive cardiomyocyte tension, and whether metformin or neuregulin-1 (NRG-1) can correct disturbed titin modifications and increased titin-based stiffness. METHODS AND RESULTS: We used cardiac biopsies from human diabetic (n=23) and nondiabetic patients (n=19), cultured rat cardiomyocytes, left ventricular tissue from apolipoprotein E-deficient mice with streptozotocin-induced diabetes mellitus (n=12-22), and ZSF1 (obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid) rats (n=5-6) and analyzed insulin-dependent signaling pathways that modulate titin phosphorylation. Titin-based passive tension was measured using permeabilized cardiomyocytes. In human diabetic hearts, we detected titin hypophosphorylation at S4099 and hyperphosphorylation at S11878, suggesting altered activity of protein kinases; cardiomyocyte passive tension was significantly increased. When applied to cultured cardiomyocytes, insulin and metformin increased titin phosphorylation at S4010, S4099, and S11878 via enhanced ERK1/2 (extracellular signal regulated kinase 1/2) and PKCα (protein kinase Cα) activity; NRG-1 application enhanced ERK1/2 activity but reduced PKCα activity. In apolipoprotein E-deficient mice, chronic treatment of streptozotocin-induced diabetes mellitus with NRG-1 corrected titin phosphorylation via increased PKG (protein kinase G) and ERK1/2 activity and reduced PKCα activity, which reversed the diabetes mellitus-associated changes in titin-based passive tension. Acute application of NRG-1 to obese ZSF1 rats with type-2 diabetes mellitus reduced end-diastolic pressure. CONCLUSIONS: Mechanistically, we found that impaired cGMP-PKG signaling and elevated PKCα activity are key modulators of titin-based cardiomyocyte stiffening in diabetic hearts. We conclude that by restoring normal kinase activities of PKG, ERK1/2, and PKCα, and by reducing cardiomyocyte passive tension, chronic NRG-1 application is a promising approach to modulate titin properties in heart failure with preserved ejection fraction associated with type-2 diabetes mellitus.

Mode-of-action of the PROPELLA concept in fulminant myocarditis.

AIMS: Haemodynamic load induces cardiac remodelling via mechano-transduction pathways, which can further trigger inflammatory responses. We hypothesized that particularly in an inflammatory disorder such as myocarditis, a therapeutic strategy is required which, in addition to providing adequate circulatory support, unloads the left ventricle, decreases cardiac wall stress, and mitigates inflammatory responses. METHODS AND RESULTS: Axial flow pumps such as the Impella systems comply with these requirements. Here, we report a potential mode-of-action of prolonged Impella support (PROPELLA concept) in fulminant myocarditis, including a decrease in cardiac immune cell presence, and integrin α1, α5, α6, α10 and ß6 expression during unloading. CONCLUSION: PROPELLA may provide benefits beyond its primary function of mechanical circulatory support in the form of additional disease-altering effects, which may contribute to enhanced myocardial recovery/remission in patients with chronic fulminant myocarditis.

The continuous heart failure spectrum: moving beyond an ejection fraction classification.

Randomized clinical trials initially used heart failure (HF) patients with low left ventricular ejection fraction (LVEF) to select study populations with high risk to enhance statistical power. However, this use of LVEF in clinical trials has led to oversimplification of the scientific view of a complex syndrome. Descriptive terms such as 'HFrEF' (HF with reduced LVEF), 'HFpEF' (HF with preserved LVEF), and more recently 'HFmrEF' (HF with mid-range LVEF), assigned on arbitrary LVEF cut-off points, have gradually arisen as separate diseases, implying distinct pathophysiologies. In this article, based on pathophysiological reasoning, we challenge the paradigm of classifying HF according to LVEF. Instead, we propose that HF is a heterogeneous syndrome in which disease progression is associated with a dynamic evolution of functional and structural changes leading to unique disease trajectories creating a spectrum of phenotypes with overlapping and distinct characteristics. Moreover, we argue that by recognizing the spectral nature of the disease a novel stratification will arise from new technologies and scientific insights that will shape the design of future trials based on deeper understanding beyond the LVEF construct alone.

Treatments targeting inotropy.

Acute heart failure (HF) and in particular, cardiogenic shock are associated with high morbidity and mortality. A therapeutic dilemma is that the use of positive inotropic agents, such as catecholamines or phosphodiesterase-inhibitors, is associated with increased mortality. Newer drugs, such as levosimendan or omecamtiv mecarbil, target sarcomeres to improve systolic function putatively without elevating intracellular Ca2+. Although meta-analyses of smaller trials suggested that levosimendan is associated with a better outcome than dobutamine, larger comparative trials failed to confirm this observation. For omecamtiv mecarbil, Phase II clinical trials suggest a favourable haemodynamic profile in patients with acute and chronic HF, and a Phase III morbidity/mortality trial in patients with chronic HF has recently begun. Here, we review the pathophysiological basis of systolic dysfunction in patients with HF and the mechanisms through which different inotropic agents improve cardiac function. Since adenosine triphosphate and reactive oxygen species production in mitochondria are intimately linked to the processes of excitation-contraction coupling, we also discuss the impact of inotropic agents on mitochondrial bioenergetics and redox regulation. Therefore, this position paper should help identify novel targets for treatments that could not only safely improve systolic and diastolic function acutely, but potentially also myocardial structure and function over a longer-term.