Cardiomuscular Biomarkers in the Diagnosis and Prognostication of Immune Checkpoint Inhibitor Myocarditis.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are approved for multiple cancers but can result in ICI-associated myocarditis, an infrequent but life-threatening condition. Elevations in cardiac biomarkers, specifically troponin-I (cTnI), troponin-T (cTnT), and creatine kinase (CK), are used for diagnosis. However, the association between temporal elevations of these biomarkers with disease trajectory and outcomes has not been established. METHODS: We analyzed the diagnostic accuracy and prognostic performances of cTnI, cTnT, and CK in patients with ICI myocarditis (n=60) through 1-year follow-up in 2 cardio-oncology units (APHP Sorbonne, Paris, France and Heidelberg, Germany). A total of 1751 (1 cTnT assay type), 920 (4 cTnI assay types), and 1191 CK sampling time points were available. Major adverse cardiomyotoxic events (MACE) were defined as heart failure, ventricular arrhythmia, atrioventricular or sinus block requiring pacemaker, respiratory muscle failure requiring mechanical ventilation, and sudden cardiac death. Diagnostic performance of cTnI and cTnT was also assessed in an international ICI myocarditis registry. RESULTS: Within 72 hours of admission, cTnT, cTnI, and CK were increased compared with upper reference limits (URLs) in 56 of 57 (98%), 37 of 42 ([88%] P=0.03 versus cTnT), and 43 of 57 ([75%] P<0.001 versus cTnT), respectively. This increased rate of positivity for cTnT (93%) versus cTnI ([64%] P<0.001) on admission was confirmed in 87 independent cases from an international registry. In the Franco-German cohort, 24 of 60 (40%) patients developed ≥1 MACE (total, 52; median time to first MACE, 5 [interquartile range, 2-16] days). The highest value of cTnT:URL within the first 72 hours of admission performed best in terms of association with MACE within 90 days (area under the curve, 0.84) than CK:URL (area under the curve, 0.70). A cTnT:URL ≥32 within 72 hours of admission was the best cut-off associated with MACE within 90 days (hazard ratio, 11.1 [95% CI, 3.2-38.0]; P<0.001), after adjustment for age and sex. cTnT was increased in all patients within 72 hours of the first MACE (23 of 23 [100%]), whereas cTnI and CK values were less than the URL in 2 of 19 (11%) and 6 of 22 (27%) of patients (P<0.001), respectively. CONCLUSIONS: cTnT is associated with MACE and is sensitive for diagnosis and surveillance in patients with ICI myocarditis. A cTnT:URL ratio <32 within 72 hours of diagnosis is associated with a subgroup at low risk for MACE. Potential differences in diagnostic and prognostic performances between cTnT and cTnI as a function of the assays used deserve further evaluation in ICI myocarditis.

Increased expression of human endogenous retrovirus K in endomyocardial biopsies from patients with cardiomyopathy - a transcriptomics meta-analysis.

Most studied, investigating transcriptional changes in myocardial biopsies focus on human genes. However, the presence and potential consequence of persistent expression of viral genes within the myocardium is unclear. The aim of the study was to analyze viral gene expression in RNAseq data from endomyocardial biopsies. The NCBI Bioproject library was screened for published projects that included bulk RNA sequencing data from endomyocardial biopsies from both healthy and diseased patients with a sample size greater than 20. Diseased patients with hypertrophic, dilated, and ischemic cardiomyopathies were included. A total of 507 patients with 507 samples from 6 bioprojects were included and mapped to the human genome (hg38). Unmappable sequences were extracted and mapped to an artificial 'super-virus' genome comprising 12,182 curated viral reference genomes. Subsequently, the sequences were reiteratively permutated and mapped again to account for randomness. In total, sequences from 68 distinct viruses were found, all of which were potentially human pathogenic. No increase in cardiotropic viruses was found in patients with dilated cardiomyopathy. However, the expression levels of the particle forming human endogenous retrovirus K were significantly increased (q < 0.0003, ANOVA). Higher expression levels were associated with increased expression in mitochondrial pathways such as oxidative phosphorylation (p < 0.0001). In Conclusion, expression of human endogenous retrovirus K is significantly increased in patients with dilated cardiomyopathy, which in turn was associated with transcriptional alterations in major cellular pathways.

Effect of SARS-CoV-2 mRNA-Vaccine on the Induction of Myocarditis in Different Murine Animal Models.

In the course of the SARS-CoV-2 pandemic, vaccination safety and risk factors of SARS-CoV-2 mRNA-vaccines were under consideration after case reports of vaccine-related side effects, such as myocarditis, which were mostly described in young men. However, there is almost no data on the risk and safety of vaccination, especially in patients who are already diagnosed with acute/chronic (autoimmune) myocarditis from other causes, such as viral infections, or as a side effect of medication and treatment. Thus, the risk and safety of these vaccines, in combination with other therapies that could induce myocarditis (e.g., immune checkpoint inhibitor (ICI) therapy), are still poorly assessable. Therefore, vaccine safety, with respect to worsening myocardial inflammation and myocardial function, was studied in an animal model of experimentally induced autoimmune myocarditis. Furthermore, it is known that ICI treatment (e.g., antibodies (abs) against PD-1, PD-L1, and CTLA-4, or a combination of those) plays an important role in the treatment of oncological patients. However, it is also known that treatment with ICIs can induce severe, life-threatening myocarditis in some patients. Genetically different A/J (most susceptible strain) and C57BL/6 (resistant strain) mice, with diverse susceptibilities for induction of experimental autoimmune myocarditis (EAM) at various age and gender, were vaccinated twice with SARS-CoV-2 mRNA-vaccine. In an additional A/J group, an autoimmune myocarditis was induced. In regard to ICIs, we tested the safety of SARS-CoV-2 vaccination in PD-1-/- mice alone, and in combination with CTLA-4 abs. Our results showed no adverse effects related to inflammation and heart function after mRNA-vaccination, independent of age, gender, and in different mouse strains susceptible for induction of experimental myocarditis. Moreover, there was no worsening effect on inflammation and cardiac function when EAM in susceptible mice was induced. However, in the experiments with vaccination and ICI treatment, we observed, in some mice, low elevation of cardiac troponins in sera, and low scores of myocardial inflammation. In sum, mRNA-vaccines are safe in a model of experimentally induced autoimmune myocarditis, but patients undergoing ICI therapy should be closely monitored when vaccinated.

Cardio-oncology imaging tools at the translational interface.

Cardiovascular imaging is an evolving component in the care of cancer patients. With improved survival following prompt cancer treatment, patients are facing increased risks of cardiovascular complications. While currently established imaging modalities are providing useful structural mechanical information, they continue to develop towards increased specificity. New modalities, emerging from basic science and oncology, are being translated, targeting earlier stages of cardiovascular disease. Besides these technical advances, matching an imaging modality with the patients' individual risk level for a specific pathological change is part of a successful imaging strategy. The choice of suitable imaging modalities and time points for specific patients will impact the cardio-oncological risk stratification during surveillance and follow-up monitoring. In addition, future imaging tools are poised to give us important insights into the underlying cardiovascular molecular pathology associated with cancer and oncological therapies. This review aims at giving an overview of the novel imaging technologies that have the potential to change cardio-oncological science and clinical practice in the near future.

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.

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.

Heart-Specific Immune Responses in an Animal Model of Autoimmune-Related Myocarditis Mitigated by an Immunoproteasome Inhibitor and Genetic Ablation.

BACKGROUND: Immune checkpoint inhibitor (ICI) therapy is often accompanied by immune-related pathology, with an increasing occurrence of high-risk ICI-related myocarditis. Understanding the mechanisms involved in this side effect could enable the development of management strategies. In mouse models, immune checkpoints, such as PD-1 (programmed cell death protein 1), control the threshold of self-antigen responses directed against cardiac TnI (troponin I). We aimed to identify how the immunoproteasome, the main proteolytic machinery in immune cells harboring 3 distinct protease activities in the LMP2 (low-molecular-weight protein 2), LMP7 (low-molecular-weight protein 7), and MECL1 (multicatalytic endopeptidase complex subunit 1) subunit, affects TnI-directed autoimmune pathology of the heart. METHODS: TnI-directed autoimmune myocarditis (TnI-AM), a CD4+ T-cell-mediated disease, was induced in mice lacking all 3 immunoproteasome subunits (triple-ip-/-) or lacking either the gene encoding LMP2 and LMP7 by immunization with a cardiac TnI peptide. Alternatively, before induction of TnI-AM or after establishment of autoimmune myocarditis, mice were treated with the immunoproteasome inhibitor ONX 0914. Immune parameters defining heart-specific autoimmunity were investigated in experimental TnI-AM and in 2 cases of ICI-related myocarditis. RESULTS: All immunoproteasome-deficient strains showed mitigated autoimmune-related cardiac pathology with less inflammation, lower proinflammatory and chemotactic cytokines, less interleukin-17 production, and reduced fibrosis formation. Protection from TnI-directed autoimmune heart pathology with improved cardiac function in LMP7-/- mice involved a changed balance between effector and regulatory CD4+ T cells in the spleen, with CD4+ T cells from LMP7-/- mice showing a higher expression of inhibitory PD-1 molecules. Blocked immunoproteasome proteolysis, by treatment of TLR2 (Toll-like receptor 2)-engaged and TLR7 (Toll-like receptor 7)/TLR8 (Toll-like receptor 8)-engaged CD14+ monocytes with ONX 0914, diminished proinflammatory cytokine responses, thereby reducing the boost for the expansion of self-reactive CD4+ T cells. Correspondingly, in mice, ONX 0914 treatment reversed cardiac autoimmune pathology, preventing the induction and progression of TnI-AM when self-reactive CD4+ T cells were primed. The autoimmune signature during experimental TnI-AM, with high immunoproteasome expression, immunoglobulin G deposition, interleukin-17 production in heart tissue, and TnI-directed humoral autoimmune responses, was also present in 2 cases of ICI-related myocarditis, demonstrating the activation of heart-specific autoimmune reactions by ICI therapy. CONCLUSIONS: By reversing heart-specific autoimmune responses, immunoproteasome inhibitors applied to a mouse model demonstrate their potential to aid in the management of autoimmune myocarditis in humans, possibly including patients with ICI-related heart-specific autoimmunity.

Epigenetic regulation of cardiac electrophysiology in atrial fibrillation: HDAC2 determines action potential duration and suppresses NRSF in cardiomyocytes.

Atrial fibrillation (AF) is associated with electrical remodeling, leading to cellular electrophysiological dysfunction and arrhythmia perpetuation. Emerging evidence suggests a key role for epigenetic mechanisms in the regulation of ion channel expression. Histone deacetylases (HDACs) control gene expression through deacetylation of histone proteins. We hypothesized that class I HDACs in complex with neuron-restrictive silencer factor (NRSF) determine atrial K+ channel expression. AF was characterized by reduced atrial HDAC2 mRNA levels and upregulation of NRSF in humans and in a pig model, with regional differences between right and left atrium. In vitro studies revealed inverse regulation of Hdac2 and Nrsf in HL-1 atrial myocytes. A direct association of HDAC2 with active regulatory elements of cardiac K+ channels was revealed by chromatin immunoprecipitation. Specific knock-down of Hdac2 and Nrsf induced alterations of K+ channel expression. Hdac2 knock-down resulted in prolongation of action potential duration (APD) in neonatal rat cardiomyocytes, whereas inactivation of Nrsf induced APD shortening. Potential AF-related triggers were recapitulated by experimental tachypacing and mechanical stretch, respectively, and exerted differential effects on the expression of class I HDACs and K+ channels in cardiomyocytes. In conclusion, HDAC2 and NRSF contribute to AF-associated remodeling of APD and K+ channel expression in cardiomyocytes via direct interaction with regulatory chromatin regions. Specific modulation of these factors may provide a starting point for the development of more individualized treatment options for atrial fibrillation.

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.

Emerging role of immune checkpoint inhibitors and their relevance for the cardiovascular system.

Immune checkpoint inhibitor (ICI) therapy induces an immune response against cancer cells. Immune checkpoint inhibitor therapy has tremendously improved the prognosis for a large number of cancers, but is associated with considerable immune-related adverse events (irAEs). Cardiovascular complications from ICI therapy occur in a modest proportion of patients, but show the highest lethality rates of all ICI-related complications. While ICI-related myocarditis is the most dangerous complication, its clinical manifestation varies, e.g., asymptomatic reduction of left ventricular function, isolated increase in cardiac troponins, and arrhythmias. This review delineates current data on cardiovascular complications of ICI therapy. The effects of ICI therapy on the cardiovascular system are classified in the context of preclinical data on the biochemical and immunological function of the immune checkpoint signaling pathways in the heart and the vascular system. Incidence, suspected pathomechanisms, typical symptoms, as well as recommended diagnostics are summarized. Current therapy recommendations for ICI-related cardiotoxicity are outlined and innovative new approaches with high potential for improving outcome in ICI-related myocarditis are delineated. A better understanding of cardiovascular complications is essential for the best possible oncocardiology care of the growing number of patients undergoing ICI therapy.

Myofilament Ca2+ sensitivity correlates with left ventricular contractility during the progression of pressure overload-induced left ventricular myocardial hypertrophy in rats.

AIM: Here we aimed at investigating the relation between left ventricular (LV) contractility and myofilament function during the development and progression of pressure overload (PO)-induced LV myocardial hypertrophy (LVH). METHODS: Abdominal aortic banding (AB) was performed to induce PO in rats for 6, 12 and 18 weeks. Sham operated animals served as controls. Structural and molecular alterations were investigated by serial echocardiography, histology, quantitative real-time PCR and western blot. LV function was assessed by pressure-volume analysis. Force measurement was carried out in permeabilized cardiomyocytes. RESULTS: AB resulted in the development of pathological LVH as indicated by increased heart weight-to-tibial length ratio, LV mass index, cardiomyocyte diameter and fetal gene expression. These alterations were already present at early stage of LVH (AB-week6). Furthermore, at more advanced stages (AB-week12, AB-week18), myocardial fibrosis and chamber dilatation were also observed. From a hemodynamic point of view, the AB-wk6 group was associated with increased LV contractility, maintained ventriculo-arterial coupling (VAC) and preserved systolic function. In the same experimental group, increased myofilament Ca2+ sensitivity (pCa50) and hyperphosphorylation of cardiac troponin-I (cTnI) at Threonine-144 was detected. In contrast, in the AB-wk12 and AB-wk18 groups, the initial augmentation of LV contractility, as well as the increased myofilament Ca2+ sensitivity and cTnI (Threonine-144) hyperphosphorylation diminished, leading to impaired VAC and reduced systolic performance. Strong correlation was found between LV contractility parameters and myofilament Ca2+-sensitivity among the study groups. CONCLUSION: Changes in myofilament Ca2+ sensitivity might underlie the alterations in LV contractility during the development and progression of PO-induced LVH.

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.

Nucleoside Diphosphate Kinase-C Suppresses cAMP Formation in Human Heart Failure.

BACKGROUND: Chronic heart failure (HF) is associated with altered signal transduction via ß-adrenoceptors and G proteins and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of patients with end-stage HF, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. METHODS: Real-time polymerase chain reaction, (far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined with immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). RESULTS: NDPK-C was essential for the formation of an NDPK-B/G protein complex. Protein and mRNA levels of NDPK-C were upregulated in end-stage human HF, in rats after long-term isoprenaline stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with isoprenaline. Isoprenaline also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to isoprenaline-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression but showed contractile dysfunction and exacerbated cardiac remodeling during long-term isoprenaline stimulation. In human end-stage HF, the complex formation between NDPK-C and Gαi2 was increased whereas the NDPK-C/Gαs interaction was decreased, producing a switch that may contribute to an NDPK-C-dependent cAMP reduction in HF. CONCLUSIONS: Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and between NDPK isoforms and G proteins. NDPK-C is a novel critical regulator of ß-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Gαs to Gαi2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.

Pressure-volume analysis reveals characteristic sex-related differences in cardiac function in a rat model of aortic banding-induced myocardial hypertrophy.

Sex differences in pressure overload (PO)-induced left ventricular (LV) myocardial hypertrophy (LVH) have been intensely investigated. Nevertheless, sex-related disparities of LV hemodynamics in LVH were not examined in detail. Therefore, we aimed to provide a detailed characterization of distinct aspects of LV function in male and female rats during different stages of LVH. Banding of the abdominal aorta (AB) was performed to induce PO for 6 or 12 wk in male and female rats. Control animals underwent sham operation. The development of LVH was followed by serial echocardiography. Cardiac function was assessed by pressure-volume analysis. Cardiomyocyte hypertrophy and fibrosis were evaluated by histology. At week 6, increased LV mass index, heart weight-to-tibial length, cardiomyocyte diameter, concentric LV geometry, and moderate interstitial fibrosis were detected in both male and female AB rats, indicating the development of an early stage of LVH. Functionally, at this time, impaired active relaxation, increased contractility, and preserved ventricular-arterial coupling were observed in the AB groups in both sexes. In contrast, at week 12, progressive deterioration of LVH-associated structural and functional alterations occurred in male but not female animals with sustained PO. Accordingly, at this later stage, LVH was associated with eccentric remodeling, exacerbated fibrosis, and increased chamber stiffness in male AB rats. Furthermore, augmented contractility declined in male but not female AB animals, resulting in contractility-afterload mismatch. Maintained contractility augmentation, preserved ventricular-arterial coupling, and better myocardial compliance in female rats contribute to sex differences in LV function during the progression of PO-induced LVH. NEW & NOTEWORTHY We investigated sex differences in pressure overload-induced left ventricular myocardial hypertrophy for the first time on the functional level by pressure-volume analysis. We found that left ventricular hypertrophy was initially characterized by prolonged active relaxation, increased contractility, and maintained ventricular-arterial coupling in both sexes. However, at a later stage, augmented contractility declined in mate but not female rats, resulting in contractility-afterload mismatch. Furthermore, in male rats, increased myocardial stiffness also contributed to hypertrophy-associated diastolic dysfunction.

Cerebral Microbleeds in Murine Amyloid Angiopathy: Natural Course and Anticoagulant Effects.

BACKGROUND AND PURPOSE: Cerebral microbleeds (CMBs) predispose patients to intracerebral hemorrhage. Preclinical models to examine the effects of antithrombotic treatments on the development of clinically overt intracerebral hemorrhage are needed. We examined the natural course of CMB development and the effects of long-term anticoagulation with warfarin or dabigatran on cerebral micro- and macrohemorrhage in mice overexpressing the APP23 (amyloid precursor protein). METHODS: Repeated susceptibility-weighted magnetic resonance imaging was performed in APP23 mice at the age of 18 and 21 months, respectively. After establishing stable long-term anticoagulation effects of warfarin and dabigatran on number and total volume of CMBs, the outcome parameters were compared with nonanticoagulated control. RESULTS: CMBs were equally located in lobar and deep brain regions, and number and total volume of CMBs increased over time. Anticoagulation with either warfarin or dabigatran did not increase CMBs in APP23 significantly. Mice treated with warfarin numerically had a higher mortality (nonanticoagulated: 31%; dabigatran: 35% versus warfarin: 55%; P=0.21). In postmortem brains of prematurely dying animals warfarin caused significantly more frequently large intracerebral hemorrhage than control and dabigatran. CONCLUSIONS: Anticoagulation with warfarin or dabigatran for 3 to 4 months does not promote the formation of CMBs in aged APP23 mice. Nevertheless, warfarin but not dabigatran is associated with a higher risk of extensive intracerebral hemorrhage, suggesting that this model may allow preclinical safety evaluation of antithrombotic therapies.

Essential role of sympathetic endothelin A receptors for adverse cardiac remodeling.

In preclinical studies, endothelin receptor A (ETA) antagonists (ETAi) attenuated the progression of heart failure (HF). However, clinical HF trials failed to demonstrate beneficial effects of ETAi. These conflicting data may be explained by the possibility that established HF drugs such as adrenergic receptor blockers interfered with the mechanism of ETAi action in clinical trials. Here we report that mice lacking ETA only in sympathetic neurons (SN-KO) showed less adverse structural remodeling and cardiac dysfunction in response to pathological pressure overload induced by transverse aortic constriction (TAC). In contrast, mice lacking ETA only in cardiomyocytes (CM-KO) were not protected. TAC led to a disturbed sympathetic nerve function as measured by cardiac norepinephrine (NE) tissue levels and [(124)I]-metaiodobenzylguanidine-PET, which was prevented in SN-KO. In a rat model of HF, ETAi improved cardiac and sympathetic nerve function. In cocultures of cardiomyocytes (CMs) and sympathetic neurons (SNs), endothelin-1 (ET1) led to a massive NE release and exaggerated CM hypertrophy compared with CM monocultures. ETA-deficient CMs gained a hypertrophic response through wild-type SNs, but ETA-deficient SNs failed to mediate exaggerated CM hypertrophy. Furthermore, ET1 mediated its effects indirectly via NE in CM-SN cocultures through adrenergic receptors and histone deacetylases, resulting in activation of the prohypertrophic transcription factor myocyte enhancer factor 2. In conclusion, sympathetic ETA amplifies ET1 effects on CMs through adrenergic signaling pathways. Thus, antiadrenergic therapies may blunt potentially beneficial effects of ETAi. Taken together, this may indicate that patients with ß blocker intolerance or disturbed sympathetic nerve function could be evaluated for a potential benefit from ETAi.

Inducible cardiomyocyte-specific deletion of CaM kinase II protects from pressure overload-induced heart failure.

CaM kinase II (CaMKII) has been suggested to drive pathological cardiac remodeling and heart failure. However, the evidence provided so far is based on inhibitory strategies using chemical compounds and peptides that also exert off-target effects and followed exclusively preventive strategies. Therefore, the aim of this study was to investigate whether specific CaMKII inhibition after the onset of cardiac stress delays or reverses maladaptive cardiac remodeling and dysfunction. Combined genetic deletion of the two redundant CaMKII genes δ and γ was induced after the onset of overt heart failure as the result of pathological pressure overload induced by transverse aortic constriction (TAC). We used two different strategies to engineer an inducible cardiomyocyte-specific CaMKIIδ/CaMKIIγ double knockout mouse model (DKO): one model bases on tamoxifen-inducible mER/Cre/mER expression under control of the cardiac-specific αMHC promoter; the other strategy bases on overexpression of Cre recombinase via cardiac-specific gene transfer through adeno-associated virus (AAV9) under control of the cardiac-specific myosin light chain promoter. Both models led to a substantial deletion of CaMKII in failing hearts. To approximate the clinical situation, CaMKII deletion was induced 3 weeks after TAC surgery. In both models of DKO, the progression of cardiac dysfunction and interstitial fibrosis could be slowed down as compared to control animals. Taken together, we show for the first time that "therapeutic" CaMKII deletion after cardiac damage is sufficient to attenuate maladaptive cardiac remodeling and to reverse signs of heart failure. These data suggest that CaMKII inhibition is a promising therapeutic approach to combat heart failure.

Oral treatment with a zinc complex of acetylsalicylic acid prevents diabetic cardiomyopathy in a rat model of type-2 diabetes: activation of the Akt pathway.

BACKGROUND: Type-2 diabetics have an increased risk of cardiomyopathy, and heart failure is a major cause of death among these patients. Growing evidence indicates that proinflammatory cytokines may induce the development of insulin resistance, and that anti-inflammatory medications may reverse this process. We investigated the effects of the oral administration of zinc and acetylsalicylic acid, in the form of bis(aspirinato)zinc(II)-complex Zn(ASA)2, on different aspects of cardiac damage in Zucker diabetic fatty (ZDF) rats, an experimental model of type-2 diabetic cardiomyopathy. METHODS: Nondiabetic control (ZL) and ZDF rats were treated orally with vehicle or Zn(ASA)2 for 24 days. At the age of 29-30 weeks, the electrical activities, left-ventricular functional parameters and left-ventricular wall thicknesses were assessed. Nitrotyrosine immunohistochemistry, TUNEL-assay, and hematoxylin-eosin staining were performed. The protein expression of the insulin-receptor and PI3K/AKT pathway were quantified by Western blot. RESULTS: Zn(ASA)2-treatment significantly decreased plasma glucose concentration in ZDF rats (39.0 ± 3.6 vs 49.4 ± 2.8 mM, P < 0.05) while serum insulin-levels were similar among the groups. Data from cardiac catheterization showed that Zn(ASA)2 normalized the increased left-ventricular diastolic stiffness (end-diastolic pressure-volume relationship: 0.064 ± 0.008 vs 0.084 ± 0.014 mmHg/µl; end-diastolic pressure: 6.5 ± 0.6 vs 7.9 ± 0.7 mmHg, P < 0.05). Furthermore, ECG-recordings revealed a restoration of prolonged QT-intervals (63 ± 3 vs 83 ± 4 ms, P < 0.05) with Zn(ASA)2. Left-ventricular wall thickness, assessed by echocardiography, did not differ among the groups. However histological examination revealed an increase in the cardiomyocytes' transverse cross-section area in ZDF compared to the ZL rats, which was significantly decreased after Zn(ASA)2-treatment. Additionally, a significant fibrotic remodeling was observed in the diabetic rats compared to ZL rats, and Zn(ASA)2-administered ZDF rats showed a similar collagen content as ZL animals. In diabetic hearts Zn(ASA)2 significantly decreased DNA-fragmentation, and nitro-oxidative stress, and up-regulated myocardial phosphorylated-AKT/AKT protein expression. Zn(ASA)2 reduced cardiomyocyte death in a cellular model of oxidative stress. Zn(ASA)2 had no effects on altered myocardial CD36, GLUT-4, and PI3K protein expression. CONCLUSIONS: We demonstrated that treatment of type-2 diabetic rats with Zn(ASA)2 reduced plasma glucose-levels and prevented diabetic cardiomyopathy. The increased myocardial AKT activation could, in part, help to explain the cardioprotective effects of Zn(ASA)2. The oral administration of Zn(ASA)2 may have therapeutic potential, aiming to prevent/treat cardiac complications in type-2 diabetic patients.