The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Myogenin and class II HDACs control neurogenic muscle atrophy by inducing E3 ubiquitin ligases.

Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an essential regulator of muscle development, controls neurogenic atrophy. Myogenin is upregulated in skeletal muscle following denervation and regulates expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, which promote muscle proteolysis and atrophy. Deletion of myogenin from adult mice diminishes expression of MuRF1 and atrogin-1 in denervated muscle and confers resistance to atrophy. Mice lacking histone deacetylases (HDACs) 4 and 5 in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation. Conversely, forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus, myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy. These findings reveal a specific pathway for muscle wasting and potential therapeutic targets for this disorder.

A network medicine approach to study comorbidities in heart failure with preserved ejection fraction.

BACKGROUND: Comorbidities are expected to impact the pathophysiology of heart failure (HF) with preserved ejection fraction (HFpEF). However, comorbidity profiles are usually reduced to a few comorbid disorders. Systems medicine approaches can model phenome-wide comorbidity profiles to improve our understanding of HFpEF and infer associated genetic profiles. METHODS: We retrospectively explored 569 comorbidities in 29,047 HF patients, including 8062 HFpEF and 6585 HF with reduced ejection fraction (HFrEF) patients from a German university hospital. We assessed differences in comorbidity profiles between HF subtypes via multiple correspondence analysis. Then, we used machine learning classifiers to identify distinctive comorbidity profiles of HFpEF and HFrEF patients. Moreover, we built a comorbidity network (HFnet) to identify the main disease clusters that summarized the phenome-wide comorbidity. Lastly, we predicted novel gene candidates for HFpEF by linking the HFnet to a multilayer gene network, integrating multiple databases. To corroborate HFpEF candidate genes, we collected transcriptomic data in a murine HFpEF model. We compared predicted genes with the murine disease signature as well as with the literature. RESULTS: We found a high degree of variance between the comorbidity profiles of HFpEF and HFrEF, while each was more similar to HFmrEF. The comorbidities present in HFpEF patients were more diverse than those in HFrEF and included neoplastic, osteologic and rheumatoid disorders. Disease communities in the HFnet captured important comorbidity concepts of HF patients which could be assigned to HF subtypes, age groups, and sex. Based on the HFpEF comorbidity profile, we predicted and recovered gene candidates, including genes involved in fibrosis (COL3A1, LOX, SMAD9, PTHL), hypertrophy (GATA5, MYH7), oxidative stress (NOS1, GSST1, XDH), and endoplasmic reticulum stress (ATF6). Finally, predicted genes were significantly overrepresented in the murine transcriptomic disease signature providing additional plausibility for their relevance. CONCLUSIONS: We applied systems medicine concepts to analyze comorbidity profiles in a HF patient cohort. We were able to identify disease clusters that helped to characterize HF patients. We derived a distinct comorbidity profile for HFpEF, which was leveraged to suggest novel candidate genes via network propagation. The identification of distinctive comorbidity profiles and candidate genes from routine clinical data provides insights that may be leveraged to improve diagnosis and identify treatment targets for HFpEF patients.

Indirect epigenetic testing identifies a diagnostic signature of cardiomyocyte DNA methylation in heart failure.

Precision-based molecular phenotyping of heart failure must overcome limited access to cardiac tissue. Although epigenetic alterations have been found to underlie pathological cardiac gene dysregulation, the clinical utility of myocardial epigenomics remains narrow owing to limited clinical access to tissue. Therefore, the current study determined whether patient plasma confers indirect phenotypic, transcriptional, and/or epigenetic alterations to ex vivo cardiomyocytes to mirror the failing human myocardium. Neonatal rat ventricular myocytes (NRVMs) and single-origin human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and were treated with blood plasma samples from patients with dilated cardiomyopathy (DCM) and donor subjects lacking history of cardiovascular disease. Following plasma treatments, NRVMs and hiPSC-CMs underwent significant hypertrophy relative to non-failing controls, as determined via automated high-content screening. Array-based DNA methylation analysis of plasma-treated hiPSC-CMs and cardiac biopsies uncovered robust, and conserved, alterations in cardiac DNA methylation, from which 100 sites were validated using an independent cohort. Among the CpG sites identified, hypo-methylation of the ATG promoter was identified as a diagnostic marker of HF, wherein cg03800765 methylation (AUC = 0.986, P < 0.0001) was found to out-perform circulating NT-proBNP levels in differentiating heart failure. Taken together, these findings support a novel approach of indirect epigenetic testing in human HF.

Loss of CASK Accelerates Heart Failure Development.

[Figure: see text].

Post-translational modifications talk and crosstalk to class IIa histone deacetylases.

Epigenetic modifications, such as histone or DNA modifications are key regulators of gene transcription and changes are often associated with maladaptive processes underlying cardiovascular disease. Epigenetic regulators therefore likely play a crucial role in cardiomyocyte homeostasis and facilitate the cellular adaption to various internal and external stimuli, responding to different intercellular and extracellular cues. Class IIa histone deacetylases are a class of epigenetic regulators that possess a myriad of post-transcriptional modification sites that modulate their activity in response to oxidative stress, altered catecholamine signalling or changes in the cellular metabolism. This review summaries the known reversible, post-translational modifications (PTMs) of class IIa histone deacetylases (HDACs) that ultimately drive transcriptional changes in homeostasis and disease. We also highlight the idea of a crosstalk of various PTMs on class IIa HDACs potentially leading to compensatory or synergistic effects on the class IIa HDAC-regulated cell behavior.

Histone deacetylase 4 deletion broadly affects cardiac epigenetic repression and regulates transcriptional susceptibility via H3K9 methylation.

Histone deacetylase 4 (HDAC4) is a member of class IIa histone deacetylases (class IIa HDACs) and is believed to possess a low intrinsic deacetylase activity. However, HDAC4 sufficiently represses distinct transcription factors (TFs) such as the myocyte enhancer factor 2 (MEF2). Transcriptional repression by HDAC4 has been suggested to be mediated by the recruitment of other chromatin-modifying enzymes, such as methyltransferases or class I histone deacetylases. However, this concept has not been investigated by an unbiased approach. Therefore, we studied the histone modifications H3K4me3, H3K9ac, H3K27ac, H3K9me2 and H3K27me3 in a genome-wide approach using HDAC4-deficient cardiomyocytes. We identified a general epigenetic shift from a 'repressive' to an 'active' status, characterized by an increase of H3K4me3, H3K9ac and H3K27ac and a decrease of H3K9me2 and H3K27me3. In HDAC4-deficient cardiomyocytes, MEF2 binding sites were considerably overrepresented in upregulated promoter regions of H3K9ac and H3K4me3. For example, we identified the promoter of Adprhl1 as a new genomic target of HDAC4 and MEF2. Overexpression of HDAC4 in cardiomyocytes was able to repress the transcription of the Adprhl1 promoter in the presence of the methyltransferase SUV39H1. On a genome-wide level, the decrease of H3K9 methylation did not change baseline expression but was associated with exercise-induced gene expression. We conclude that HDAC4, on the one hand, associates with activating histone modifications, such as H3K4me3 and H3K9ac. A functional consequence, on the other hand, requires an indirect regulation of H3K9me2. H3K9 hypomethylation in HDAC4 target genes ('first hit') plus a 'second hit' (e.g., exercise) determines the transcriptional response.

Deep phenotyping of two preclinical mouse models and a cohort of RBM20 mutation carriers reveals no sex-dependent disease severity in RBM20 cardiomyopathy.

RBM20 cardiomyopathy is an arrhythmogenic form of dilated cardiomyopathy caused by mutations in the splicing factor RBM20. A recent study found a more severe phenotype in male patients with RBM20 cardiomyopathy patients than in female patients. Here, we aim to determine sex differences in an animal model of RBM20 cardiomyopathy and investigate potential underlying mechanisms. In addition, we aim to determine sex and gender differences in clinical parameters in a novel RBM20 cardiomyopathy patient cohort. We characterized an Rbm20 knockout (KO) mouse model, and show that splicing of key RBM20 targets, cardiac function, and arrhythmia susceptibility do not differ between sexes. Next, we performed deep phenotyping of these mice, and show that male and female Rbm20-KO mice possess transcriptomic and phosphoproteomic differences. Hypothesizing that these differences may influence the heart's ability to compensate for stress, we exposed Rbm20-KO mice to acute catecholaminergic stimulation and again found no functional differences. We also replicate the lack of functional differences in a mouse model with the Rbm20-R636Q mutation. Lastly, we present a patient cohort of 33 RBM20 cardiomyopathy patients and show that these patients do not possess sex and gender differences in disease severity. Current mouse models of RBM20 cardiomyopathy show more pronounced changes in gene expression and phosphorylation of cardiac proteins in male mice, but no sex differences in cardiac morphology and function. Moreover, other than reported before, male RBM20 cardiomyopathy patients do not present with worse cardiac function in a patient cohort from Germany and the Netherlands.NEW & NOTEWORTHY Optimal management of the cardiac disease is increasingly personalized, partly because of differences in outcomes between sexes. RBM20 cardiomyopathy has been described to be more severe in male patients, and this carries the risk that male patients are more scrutinized in the clinic than female patients. Our findings do not support this observation and suggest that treatment should not differ between male and female RBM20 cardiomyopathy patients, but instead should focus on the underlying disease mechanism.

Chronic isoprenaline/phenylephrine vs. exclusive isoprenaline stimulation in mice: critical contribution of alpha1-adrenoceptors to early cardiac stress responses.

Hyperactivity of the sympathetic nervous system is a major driver of cardiac remodeling, exerting its effects through both α-, and ß-adrenoceptors (α-, ß-ARs). As the relative contribution of subtype α1-AR to cardiac stress responses remains poorly investigated, we subjected mice to either subcutaneous perfusion with the ß-AR agonist isoprenaline (ISO, 30 mg/kg × day) or to a combination of ISO and the stable α1-AR agonist phenylephrine (ISO/PE, 30 mg/kg × day each). Telemetry analysis revealed similar hemodynamic responses under both ISO and ISO/PE treatment i.e., permanently increased heart rates and only transient decreases in mean blood pressure during the first 24 h. Echocardiography and single cell analysis after 1 week of exposure showed that ISO/PE-, but not ISO-treated animals established α1-AR-mediated inotropic responsiveness to acute adrenergic stimulation. Morphologically, additional PE perfusion limited concentric cardiomyocyte growth and enhanced cardiac collagen deposition during 7 days of treatment. Time-course analysis demonstrated a diverging development in transcriptional patterns at day 4 of treatment i.e., increased expression of selected marker genes Xirp2, Nppa, Tgfb1, Col1a1, Postn under chronic ISO/PE treatment which was either less pronounced or absent in the ISO group. Transcriptome analyses at day 4 via RNA sequencing demonstrated that additional PE treatment caused a marked upregulation of genes allocated to extracellular matrix and fiber organization along with a more pronounced downregulation of genes involved in metabolic processes, muscle adaptation and cardiac electrophysiology. Consistently, transcriptome changes under ISO/PE challenge more effectively recapitulated early transcriptional alterations in pressure overload-induced experimental heart failure and in human hypertrophic cardiomyopathy.

Forebrain corticosteroid receptors promote post-myocardial infarction depression and mortality.

Myocardial infarction (MI) with subsequent depression is associated with increased cardiac mortality. Impaired central mineralocorticoid (MR) and glucocorticoid receptor (GR) equilibrium has been suggested as a key mechanism in the pathogenesis of human depression. Here, we investigate if deficient central MR/GR signaling is causative for a poor outcome after MI in mice. Mice with an inducible forebrain-specific MR/GR knockout (MR/GR-KO) underwent baseline and follow-up echocardiography every 2 weeks after MI or sham operation. Behavioral testing at 4 weeks confirmed significant depressive-like behavior and, strikingly, a higher mortality after MI, while cardiac function and myocardial damage remained unaffected. Telemetry revealed cardiac autonomic imbalance with marked bradycardia and ventricular tachycardia (VT) upon MI in MR/GR-KO. Mechanistically, we found a higher responsiveness to atropine, pointing to impaired parasympathetic tone of 'depressive' mice after MI. Serum corticosterone levels were increased but-in line with the higher vagal tone-plasma and cardiac catecholamines were decreased. MR/GR deficiency in the forebrain led to significant depressive-like behavior and a higher mortality after MI. This was accompanied by increased vagal tone, depleted catecholaminergic compensatory capacity and VTs. Thus, limbic MR/GR disequilibrium may contribute to the impaired outcome of depressive patients after MI and possibly explain the lack of anti-depressive treatment benefit.

CaM kinase II regulates cardiac hemoglobin expression through histone phosphorylation upon sympathetic activation.

Sympathetic activation of ß-adrenoreceptors (ß-AR) represents a hallmark in the development of heart failure (HF). However, little is known about the underlying mechanisms of gene regulation. In human ventricular myocardium from patients with end-stage HF, we found high levels of phosphorylated histone 3 at serine-28 (H3S28p). H3S28p was increased by inhibition of the catecholamine-sensitive protein phosphatase 1 and decreased by ß-blocker pretreatment. By a series of in vitro and in vivo experiments, we show that the ß-AR downstream protein kinase CaM kinase II (CaMKII) directly binds and phosphorylates H3S28. Whereas, in CaMKII-deficient myocytes, acute catecholaminergic stimulation resulted in some degree of H3S28p, sustained catecholaminergic stimulation almost entirely failed to induce H3S28p. Genome-wide analysis of CaMKII-mediated H3S28p in response to chronic ß-AR stress by chromatin immunoprecipitation followed by massive genomic sequencing led to the identification of CaMKII-dependent H3S28p target genes. Forty percent of differentially H3S28p-enriched genomic regions were associated with differential, mostly increased expression of the nearest genes, pointing to CaMKII-dependent H3S28p as an activating histone mark. Remarkably, the adult hemoglobin genes showed an H3S28p enrichment close to their transcriptional start or end sites, which was associated with increased messenger RNA and protein expression. In summary, we demonstrate that chronic ß-AR activation leads to CaMKII-mediated H3S28p in cardiomyocytes. Thus, H3S28p-dependent changes may play an unexpected role for cardiac hemoglobin regulation in the context of sympathetic activation. These data also imply that CaMKII may be a yet unrecognized stress-responsive regulator of hematopoesis.

Diabetes changes gene expression but not DNA methylation in cardiac cells.

BACKGROUND: Diabetes mellitus is a worldwide epidemic that causes high mortality due to cardiovascular complications, in particular heart failure. Diabetes is associated with profound pathophysiological changes in the heart. The aim of this study was to investigate the impact of diabetes on gene expression and DNA methylation in cardiac cells. METHODS AND RESULTS: Transcriptome analysis of heart tissue from mice with streptozotocin-induced diabetes revealed only 39 genes regulated, whereas cell type-specific analysis of the diabetic heart was more sensitive and more specific than heart tissue analysis and revealed a total of 3205 differentially regulated genes in five cell types. Whole genome DNA methylation analysis with basepair resolution of distinct cardiac cell types identified highly specific DNA methylation signatures of genic and regulatory regions. Interestingly, despite marked changes in gene expression, DNA methylation remained stable in streptozotocin-induced diabetes. Integrated analysis of cell type-specific gene expression enabled us to assign the particular contribution of single cell types to the pathophysiology of the diabetic heart. Finally, analysis of gene regulation revealed ligand-receptor pairs as potential mediators of heterocellular interaction in the diabetic heart, with fibroblasts and monocytes showing the highest degree of interaction. CONCLUSION: In summary, cell type-specific analysis reveals differentially regulated gene programs that are associated with distinct biological processes in diabetes. Interestingly, despite these changes in gene expression, cell type-specific DNA methylation signatures of genic and regulatory regions remain stable in diabetes. Analysis of heterocellular interactions in the diabetic heart suggest that the interplay between fibroblasts and monocytes is of pivotal importance.

Secretome Analysis of Cardiomyocytes Identifies PCSK6 (Proprotein Convertase Subtilisin/Kexin Type 6) as a Novel Player in Cardiac Remodeling After Myocardial Infarction.

BACKGROUND: Acute occlusion of a coronary artery results in swift tissue necrosis. Bordering areas of the infarcted myocardium can also experience impaired blood supply and reduced oxygen delivery, leading to altered metabolic and mechanical processes. Although transcriptional changes in hypoxic cardiomyocytes are well studied, little is known about the proteins that are actively secreted from these cells. METHODS: We established a novel secretome analysis of cardiomyocytes by combining stable isotope labeling and click chemistry with subsequent mass spectrometry analysis. Further functional validation experiments included ELISA measurement of human samples, murine left anterior descending coronary artery ligation, and adeno-associated virus 9-mediated in vivo overexpression in mice. RESULTS: The presented approach is feasible for analysis of the secretome of primary cardiomyocytes without serum starvation. A total of 1026 proteins were identified to be secreted within 24 hours, indicating a 5-fold increase in detection compared with former approaches. Among them, a variety of proteins have not yet been explored in the context of cardiovascular pathologies. One of the secreted factors most strongly upregulated upon hypoxia was PCSK6 (proprotein convertase subtilisin/kexin type 6). Validation experiments revealed an increase of PCSK6 on mRNA and protein level in hypoxic cardiomyocytes. PCSK6 expression was elevated in hearts of mice after 3 days of ligation of the left anterior descending artery, a finding confirmed by immunohistochemistry. ELISA measurements in human serum also indicate distinct kinetics for PCSK6 in patients with acute myocardial infarction, with a peak on postinfarction day 3. Transfer of PCSK6-depleted cardiomyocyte secretome resulted in decreased expression of collagen I and III in fibroblasts compared with control treated cells, and small interfering RNA-mediated knockdown of PCSK6 in cardiomyocytes impacted transforming growth factor-ß activation and SMAD3 (mothers against decapentaplegic homolog 3) translocation in fibroblasts. An adeno-associated virus 9-mediated, cardiomyocyte-specific overexpression of PCSK6 in mice resulted in increased collagen expression and cardiac fibrosis, as well as decreased left ventricular function, after myocardial infarction. CONCLUSIONS: A novel mass spectrometry-based approach allows investigation of the secretome of primary cardiomyocytes. Analysis of hypoxia-induced secretion led to the identification of PCSK6 as being crucially involved in cardiac remodeling after acute myocardial infarction.

Monitoring Cell-Type-Specific Gene Expression Using Ribosome Profiling In Vivo During Cardiac Hemodynamic Stress.

RATIONALE: Gene expression profiles have been mainly determined by analysis of transcript abundance. However, these analyses cannot capture posttranscriptional gene expression control at the level of translation, which is a key step in the regulation of gene expression, as evidenced by the fact that transcript levels often poorly correlate with protein levels. Furthermore, genome-wide transcript profiling of distinct cell types is challenging due to the fact that lysates from tissues always represent a mixture of cells. OBJECTIVES: This study aimed to develop a new experimental method that overcomes both limitations and to apply this method to perform a genome-wide analysis of gene expression on the translational level in response to pressure overload. METHODS AND RESULTS: By combining ribosome profiling (Ribo-seq) with a ribosome-tagging approach (Ribo-tag), it was possible to determine the translated transcriptome in specific cell types from the heart. After pressure overload, we monitored the cardiac myocyte translatome by purifying tagged cardiac myocyte ribosomes from cardiac lysates and subjecting the ribosome-protected mRNA fragments to deep sequencing. We identified subsets of mRNAs that are regulated at the translational level and found that translational control determines early changes in gene expression in response to cardiac stress in cardiac myocytes. Translationally controlled transcripts are associated with specific biological processes related to translation, protein quality control, and metabolism. Mechanistically, Ribo-seq allowed for the identification of upstream open reading frames in transcripts, which we predict to be important regulators of translation. CONCLUSIONS: This method has the potential to (1) provide a new tool for studying cell-specific gene expression at the level of translation in tissues, (2) reveal new therapeutic targets to prevent cellular remodeling, and (3) trigger follow-up studies that address both, the molecular mechanisms involved in the posttranscriptional control of gene expression in cardiac cells, and the protective functions of proteins expressed in response to cellular stress.

Cyclic AMP represses pathological MEF2 activation by myocyte-specific hypo-phosphorylation of HDAC5.

Class IIa histone deacetylases (HDACs) critically regulate cardiac function through the repression of the activity of myocyte enhancer factor 2 (MEF2)-dependent gene programs. Protein kinase D (PKD) and Ca2+/Calmodulin-dependent kinase II (CaMKII) activate MEF2 by phosphorylating distinct HDAC isoforms and thereby creating 14-3-3 binding sites for nucleo-cytoplasmic shuttling. Recently, it has been shown that this process is counteracted by cyclic AMP (cAMP)-dependent signaling. Here, we investigated the specific mechanisms of how cAMP-dependent signaling regulates distinct HDAC isoforms and determined their relative contributions to the protection from pathological MEF2 activation. We found that cAMP is sufficient to induce nuclear retention and to blunt phosphorylation of the 14-3-3 binding sites of HDAC5 (Ser259/498) and HDAC9 (Ser218/448) but not HDAC4 (Ser246/467/632). These regulatory events could be observed only in cardiomyocytes and myocyte-like cells but not in non-myocytes, pointing to an indirect myocyte-specific mode of action. Consistent with one previous report, we found that blunted phosphorylation of HDAC5 and HDAC9 was mediated by protein kinase A (PKA)-dependent inhibition of PKD. However, we show by the use of neonatal cardiomyocytes derived from genetic HDAC mouse models that endogenous HDAC5 but not HDAC9 contributes specifically to the repression of endogenous MEF2 activity. HDAC4 contributed significantly to the repression of MEF2 activity but based on the mechanistic findings of this study combined with previous results we attribute this to PKA-dependent proteolysis of HDAC4. Consistently, cAMP-induced repression of agonist-driven cellular hypertrophy was blunted in cardiomyocytes deficient for both HDAC5 and HDAC4. In conclusion, cAMP inhibits MEF2 through both nuclear accumulation of hypo-phosphorylated HDAC5 and through a distinct HDAC4-dependent mechanism.

CaMKII does not control mitochondrial Ca2+ uptake in cardiac myocytes.

KEY POINTS: Mitochondrial Ca2+ uptake stimulates the Krebs cycle to regenerate the reduced forms of pyridine nucleotides (NADH, NADPH and FADH2 ) required for ATP production and reactive oxygen species (ROS) elimination. Ca2+ /calmodulin-dependent protein kinase II (CaMKII) has been proposed to regulate mitochondrial Ca2+ uptake via mitochondrial Ca2+ uniporter phosphorylation. We used two mouse models with either global deletion of CaMKIIδ (CaMKIIδ knockout) or cardiomyocyte-specific deletion of CaMKIIδ and γ (CaMKIIδ/γ double knockout) to interrogate whether CaMKII controls mitochondrial Ca2+ uptake in isolated mitochondria and during ß-adrenergic stimulation in cardiac myocytes. CaMKIIδ/γ did not control Ca2+ uptake, respiration or ROS emission in isolated cardiac mitochondria, nor in isolated cardiac myocytes, during ß-adrenergic stimulation and pacing. The results of the present study do not support a relevant role of CaMKII for mitochondrial Ca2+ uptake in cardiac myocytes under physiological conditions. ABSTRACT: Mitochondria are the main source of ATP and reactive oxygen species (ROS) in cardiac myocytes. Furthermore, activation of the mitochondrial permeability transition pore (mPTP) induces programmed cell death. These processes are essentially controlled by Ca2+ , which is taken up into mitochondria via the mitochondrial Ca2+ uniporter (MCU). It was recently proposed that Ca2+ /calmodulin-dependent protein kinase II (CaMKII) regulates Ca2+ uptake by interacting with the MCU, thereby affecting mPTP activation and programmed cell death. In the present study, we investigated the role of CaMKII under physiological conditions in which mitochondrial Ca2+ uptake matches energy supply to the demand of cardiac myocytes. Accordingly, we measured mitochondrial Ca2+ uptake in isolated mitochondria and cardiac myocytes harvested from cardiomyocyte-specific CaMKII δ and γ double knockout (KO) (CaMKIIδ/γ DKO) and global CaMKIIδ KO mice. To simulate a physiological workload increase, cardiac myocytes were subjected to ß-adrenergic stimulation (by isoproterenol superfusion) and an increase in stimulation frequency (from 0.5 to 5 Hz). No differences in mitochondrial Ca2+ accumulation were detected in isolated mitochondria or cardiac myocytes from both CaMKII KO models compared to wild-type littermates. Mitochondrial redox state and ROS production were unchanged in CaMKIIδ/γ DKO, whereas we observed a mild oxidation of mitochondrial redox state and an increase in H2 O2 emission from CaMKIIδ KO cardiac myocytes exposed to an increase in workload. In conclusion, the results obtained in the present study do not support the regulation of mitochondrial Ca2+ uptake via the MCU or mPTP activation by CaMKII in cardiac myocytes under physiological conditions.

O-GlcNAcylation of Histone Deacetylase 4 Protects the Diabetic Heart From Failure.

BACKGROUND: Worldwide, diabetes mellitus and heart failure represent frequent comorbidities with high socioeconomic impact and steadily growing incidence, calling for a better understanding of how diabetic metabolism promotes cardiac dysfunction. Paradoxically, some glucose-lowering drugs have been shown to worsen heart failure, raising the question of how glucose mediates protective versus detrimental cardiac signaling. Here, we identified a histone deacetylase 4 (HDAC4) subdomain as a molecular checkpoint of adaptive and maladaptive signaling in the diabetic heart. METHODS: A conditional HDAC4 allele was used to delete HDAC4 specifically in cardiomyocytes (HDAC4-knockout). Mice were subjected to diabetes mellitus either by streptozotocin injections (type 1 diabetes mellitus model) or by crossing into mice carrying a leptin receptor mutation (db/db; type 2 diabetes mellitus model) and monitored for remodeling and cardiac function. Effects of glucose and the posttranslational modification by ß-linked N-acetylglucosamine (O-GlcNAc) on HDAC4 were investigated in vivo and in vitro by biochemical and cellular assays. RESULTS: We show that the cardio-protective N-terminal proteolytic fragment of HDAC4 is enhanced in vivo in patients with diabetes mellitus and mouse models, as well as in vitro under high-glucose and high-O-GlcNAc conditions. HDAC4-knockout mice develop heart failure in models of type 1 and type 2 diabetes mellitus, whereas wild-type mice do not develop clear signs of heart failure, indicating that HDAC4 protects the diabetic heart. Reexpression of the N-terminal fragment of HDAC4 prevents HDAC4-dependent diabetic cardiomyopathy. Mechanistically, the posttranslational modification of HDAC4 at serine (Ser)-642 by O-GlcNAcylation is an essential step for production of the N-terminal fragment of HDAC4, which was attenuated by Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation at Ser-632. Preventing O-GlcNAcylation at Ser-642 not only entirely precluded production of the N-terminal fragment of HDAC4 but also promoted Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation at Ser-632, pointing to a mutual posttranslational modification cross talk of (cardio-detrimental) phosphorylation at Ser-632 and (cardio-protective) O-GlcNAcylation at Ser-642. CONCLUSIONS: In this study, we found that O-GlcNAcylation of HDAC4 at Ser-642 is cardio-protective in diabetes mellitus and counteracts pathological Ca2+/calmodulin-dependent protein kinase II signaling. We introduce a molecular model explaining how diabetic metabolism possesses important cardio-protective features besides its known detrimental effects. A deeper understanding of the here-described posttranslational modification cross talk may lay the groundwork for the development of specific therapeutic concepts to treat heart failure in the context of diabetes mellitus.

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.

RBM20 Mutations Induce an Arrhythmogenic Dilated Cardiomyopathy Related to Disturbed Calcium Handling.

BACKGROUND: Mutations in RBM20 (RNA-binding motif protein 20) cause a clinically aggressive form of dilated cardiomyopathy, with an increased risk of malignant ventricular arrhythmias. RBM20 is a splicing factor that targets multiple pivotal cardiac genes, such as Titin (TTN) and CAMK2D (calcium/calmodulin-dependent kinase II delta). Aberrant TTN splicing is thought to be the main determinant of RBM20-induced dilated cardiomyopathy, but is not likely to explain the increased risk of arrhythmias. Here, we investigated the extent to which RBM20 mutation carriers have an increased risk of arrhythmias and explore the underlying molecular mechanism. METHODS: We compared clinical characteristics of RBM20 and TTN mutation carriers and used our previously generated Rbm20 knockout (KO) mice to investigate downstream effects of Rbm20-dependent splicing. Cellular electrophysiology and Ca2+ measurements were performed on isolated cardiomyocytes from Rbm20 KO mice to determine the intracellular consequences of reduced Rbm20 levels. RESULTS: Sustained ventricular arrhythmias were more frequent in human RBM20 mutation carriers than in TTN mutation carriers (44% versus 5%, respectively, P=0.006). Splicing events that affected Ca2+- and ion-handling genes were enriched in Rbm20 KO mice, most notably in the genes CamkIIδ and RyR2. Aberrant splicing of CamkIIδ in Rbm20 KO mice resulted in a remarkable shift of CamkIIδ toward the δ-A isoform that is known to activate the L-type Ca2+ current ( ICa,L). In line with this, we found an increased ICa,L, intracellular Ca2+ overload and increased sarcoplasmic reticulum Ca2+ content in Rbm20 KO myocytes. In addition, not only complete loss of Rbm20, but also heterozygous loss of Rbm20 increased spontaneous sarcoplasmic reticulum Ca2+ releases, which could be attenuated by treatment with the ICa,L antagonist verapamil. CONCLUSIONS: We show that loss of Rbm20 disturbs Ca2+ handling and leads to more proarrhythmic Ca2+ releases from the sarcoplasmic reticulum. Patients that carry a pathogenic RBM20 mutation have more ventricular arrhythmias despite a similar left ventricular function, in comparison with patients with a TTN mutation. Our experimental data suggest that RBM20 mutation carriers may benefit from treatment with an ICa,L blocker to reduce their arrhythmia burden.

Inhibition of Endothelial Notch Signaling Impairs Fatty Acid Transport and Leads to Metabolic and Vascular Remodeling of the Adult Heart.

BACKGROUND: Nutrients are transported through endothelial cells before being metabolized in muscle cells. However, little is known about the regulation of endothelial transport processes. Notch signaling is a critical regulator of metabolism and angiogenesis during development. Here, we studied how genetic and pharmacological manipulation of endothelial Notch signaling in adult mice affects endothelial fatty acid transport, cardiac angiogenesis, and heart function. METHODS: Endothelial-specific Notch inhibition was achieved by conditional genetic inactivation of Rbp-jκ in adult mice to analyze fatty acid metabolism and heart function. Wild-type mice were treated with neutralizing antibodies against the Notch ligand Delta-like 4. Fatty acid transport was studied in cultured endothelial cells and transgenic mice. RESULTS: Treatment of wild-type mice with Delta-like 4 neutralizing antibodies for 8 weeks impaired fractional shortening and ejection fraction in the majority of mice. Inhibition of Notch signaling specifically in the endothelium of adult mice by genetic ablation of Rbp-jκ caused heart hypertrophy and failure. Impaired heart function was preceded by alterations in fatty acid metabolism and an increase in cardiac blood vessel density. Endothelial Notch signaling controlled the expression of endothelial lipase, Angptl4, CD36, and Fabp4, which are all needed for fatty acid transport across the vessel wall. In endothelial-specific Rbp-jκ-mutant mice, lipase activity and transendothelial transport of long-chain fatty acids to muscle cells were impaired. In turn, lipids accumulated in the plasma and liver. The attenuated supply of cardiomyocytes with long-chain fatty acids was accompanied by higher glucose uptake, increased concentration of glycolysis intermediates, and mTOR-S6K signaling. Treatment with the mTOR inhibitor rapamycin or displacing glucose as cardiac substrate by feeding a ketogenic diet prolonged the survival of endothelial-specific Rbp-jκ-deficient mice. CONCLUSIONS: This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function.