Friederike Cuello

Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology.

More than 350 individual MYPBC3 mutations have been identified in patients with inherited hypertrophic cardiomyopathy (HCM), thus representing 40–50% of all HCM mutations, making it the most frequently mutated gene in HCM. HCM is considered a disease of the sarcomere and is characterized by left ventricular hypertrophy, myocyte disarray and diastolic dysfunction. MYBPC3 encodes for the thick filament associated protein cardiac myosin-binding protein C (cMyBP-C), a signaling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of contraction and relaxation. This review aims to provide a succinct overview of how mutations in MYBPC3 are considered to affect the physiological function of cMyBP-C, thus causing the deleterious consequences observed inHCM patients. Importantly, recent advances to causally treat HCM by repairing MYBPC3 mutations by gene therapy are discussed here, providing a promising alternative to heart transplantation for patients with a fatal form of neonatal cardiomyopathy due to bi-allelic truncating MYBPC3 mutations.

Ranolazine antagonizes catecholamine-induced dysfunction in isolated cardiomyocytes, but lacks long-term therapeutic effects in vivo in a mouse model of hypertrophic cardiomyopathy

AIMSnHypertrophic cardiomyopathy (HCM) is often accompanied by increased myofilament Ca(2+) sensitivity and diastolic dysfunction. Recent findings indicate increased late Na(+) current density in human HCM cardiomyocytes. Since ranolazine has the potential to decrease myofilament Ca(2+) sensitivity and late Na(+) current, we investigated its effects in an Mybpc3-targeted knock-in (KI) mouse model of HCM.nnnMETHODS AND RESULTSnUnloaded sarcomere shortening and Ca(2+) transients were measured in KI and wild-type (WT) cardiomyocytes. Measurements were performed at baseline (1 Hz) and under increased workload (30 nM isoprenaline (ISO), 5 Hz) in the absence or presence of 10 µM ranolazine. KI myocytes showed shorter diastolic sarcomere length at baseline, stronger inotropic response to ISO, and drastic drop of diastolic sarcomere length under increased workload. Ranolazine attenuated ISO responses in WT and KI cells and prevented workload-induced diastolic failure in KI. Late Na(+) current density was diminished and insensitive to ranolazine in KI cardiomyocytes. Ca(2+) sensitivity of skinned KI trabeculae was slightly decreased by ranolazine. Phosphorylation analysis of cAMP-dependent protein kinase A-target proteins and ISO concentration-response measurements on muscle strips indicated antagonism at β-adrenoceptors with 10 µM ranolazine shifting the ISO response by 0.6 log units. Six-month treatment with ranolazine (plasma level >20 µM) demonstrated a β-blocking effect, but did not reverse cardiac hypertrophy or dysfunction in KI mice.nnnCONCLUSIONnRanolazine improved tolerance to high workload in mouse HCM cardiomyocytes, not by blocking late Na(+) current, but by antagonizing β-adrenergic stimulation and slightly desensitizing myofilaments to Ca(2+). This effect did not translate in therapeutic efficacy in vivo.

Heart failure-specific changes in protein kinase signalling

Among the myriad of molecular alterations occurring in heart failure development, aggravation of the disease is often attributed to global or local changes in protein kinase activity, thus making protein kinases attractive targets for therapeutic intervention. Since protein kinases do not only have maladaptive roles, but also contribute to the physiological integrity of cells, it is a challenging task to circumvent undesired inhibition of protein kinase activity. Identification of posttranslational modifications and/or protein-protein interactions that are exclusively apparent under pathophysiological conditions provides exciting information for alternative non-kinase inhibitory treatment strategies that eliminate maladaptive functions of a protein kinase, but preserve the beneficial ones. Here, we focus on the disease-specific regulation of a number of protein kinases, namely, Ca2+/calmodulin-dependent protein kinase II isoform δ (CaMKIIδ), G protein-coupled receptor kinase 2 (GRK2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein kinase D (PKD) and protein kinase C isoform β2 (PKCβ2), which are embedded in complex signal transduction pathways implicated in heart failure development, and discuss potential avenues for novel treatment strategies to combat heart disease.

Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue

Introduction Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism. Methods and Results We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy). Conclusion This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux.

S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure

Cardiac myosin‐binding protein C (cMyBP‐C) regulates actin‐myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP‐C phosphorylation. In our study, reduced site‐specific cMyBP‐C phosphorylation coincided with increased S‐glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease‐specific S‐glutathiolation sites in cMyBP‐C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1‐M‐C2 was identified and showed enhanced S‐glutathiolation in patients. In vitro S‐glutathiolation of recombinant cMyBP‐C C1‐M‐C2 occurred predominantly at Cys249, which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP‐C S‐glutathiolation, which functionally decelerated the kinetics of Ca2+‐activated force development in ventricular myocytes from wild‐type, but not those from Mybpc3‐targeted knockout mice. These oxidation events abrogate protein kinase‐mediated phosphorylation of cMyBP‐C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart failure.—Stathopoulou, K., Wittig, I., Heidler, J., Piasecki, A., Richter, F., Diering, S., van der Velden, J., Buck, F., Donzelli, S., Schröder, E., Wijnker, P. J. M., Voigt, N., Dobrev, D., Sadayappan, S., Eschenhagen, T., Carrier, L., Eaton, P., Cuello, F. S‐glutathiolation impairs phosphoregulation and function of cardiac myosin‐binding protein C in human heart failure. FASEB J. 30, 1849–1864 (2016). www.fasebj.org

Inhibition of cardiac CaMKII to cure heart failure: step by step towards translation?

During the past decade, calcium/calmodulin-dependent protein kinase II (CaMKII) has emerged as a central culprit in the development of cardiac arrhythmia and heart failure. This has been supported by a plethora of studies using transgenic mouse models and pharmacological CaMKII inhibitors and peptides. However, the final proof that CaMKII inhibition improves dysfunction of a failing heart is still pending. In this issue, Kreusser et al. [19] demonstrated that knockdown of the two key cardiac CaMKII isoforms after the onset of hemodynamic stress succeeds to reverse maladaptive cardiac remodeling processes. Their study is an important step in translating CaMKII inhibition from bench to bedside for heart failure therapy. CaMKII is a multifunctional protein kinase that plays a pivotal role in cardiac (patho) physiology [27, 40]. It represents a nodal point in the regulation of intracellular Ca handling, ion channels and gene transcription. As the description ‘‘multifunctional’’ and ‘‘nodal point’’ implies, this kinase is intertwined into a complex cellular signaling network and thus tricky to manipulate therapeutically: CaMKII activity is regulated by posttranslational modifications that allow maintenance of kinase activity independently of Ca/calmodulin-binding. Amongst those autophosphorylation [16, 26, 45], oxidation [2, 7, 13, 32, 45], O-linked N-acetylglucosamination [9] and S-nitrosylation [8, 12] have been described to date. CaMKII has various cellular targets in Ca homeostasis some of which are the ryanodine receptor [26, 34, 39, 41], phospholamban [5, 18] and cardiac myosin-binding protein C [37]. Furthermore, CaMKII impacts on L-type Ca channel (LTCC) currents and LTCC expression [40], on expression of the Na/Ca exchanger [10, 23] and the sarcoplasmic reticulum Ca ATPase 2a (SERCA2a) [5, 23, 42] as well as on gene transcription via the regulation of calcineurin and class II histone deacetylase isoforms [3, 24]. CaMKII is ubiquitously expressed, with a and b as predominant isoforms in the brain, where they are important for neuronal function and cognitive memory. CaMKIIc and d are the key isoforms expressed in the heart. Particular attention has been paid to two CaMKIId splice variants in the heart, CaMKIIdB and CaMKIIdC. CaMKIIdB has an 11 amino acid nuclear localization sequence that is absent in CaMKIIdC. Studies performed in splice-variant-specific knockout mouse models have attributed a protective functional role to CaMKIIdB. Thus, cellular localization seems to participate in CaMKII isoform-specific pathophysiological roles [4, 5, 28, 44]. Despite of the physiological importance of CaMKII, for e.g,. excitation–contraction coupling, isoproterenol-induced This comment refers to the article available at doi:10.1007/s00395016-0581-2.

Cysteine-Based Redox Sensing and Its Role in Signaling by Cyclic Nucleotide-Dependent Kinases in the Cardiovascular System

Oxidant molecules are produced in biological systems and historically have been considered causal mediators of damage and disease. While oxidants may contribute to the pathogenesis of disease, evidence continues to emerge that shows these species also play important regulatory roles in health. A major mechanism of oxidant sensing and signaling involves their reaction with reactive cysteine thiols within proteins, inducing oxidative posttranslational modifications that can couple to altered function to enable homeostatic regulation. Protein kinase A and protein kinase G are regulated by oxidants in this way, and this review focuses on our molecular-level understanding of these events and their role in regulating cardiovascular physiology during health and disease.

Oxidation of cardiac myofilament proteins: Priming for dysfunction?

Oxidants are produced endogenously and can react with and thereby post-translationally modify target proteins. They have been implicated in the redox regulation of signal transduction pathways conferring protection, but also in mediating oxidative stress and causing damage. The difference is that in scenarios of injury the amount of oxidants generated is higher and/or the duration of oxidant exposure sustained. In the cardiovascular system, oxidants are important for blood pressure homeostasis, for unperturbed cardiac function and also contribute to the observed protection during ischemic preconditioning. In contrast, oxidative stress accompanies all major cardiovascular pathologies and has been attributed to mediate contractile dysfunction in part by inducing oxidative modifications in myofilament proteins. However, the proportion to which oxidative modifications of contractile proteins are beneficial or causatively mediate disease progression needs to be carefully reconsidered. These antithetical aspects will be discussed in this review with special focus on direct oxidative post-translational modifications of myofilament proteins that have been described to occur in vivo and to regulate actin-myosin interactions in the cardiac myocyte sarcomere, the methodologies for detection of oxidative post-translational modifications in target proteins and the feasibility of antioxidant therapy strategies as a potential treatment for cardiac disorders.

Oxidant sensor in the cGMP-binding pocket of PKGIα regulates nitroxyl-mediated kinase activity

Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli’s salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGIα that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGIα. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGIα. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGIα. Intradisulfide formation in PKGIα correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGIα, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.