Prdm16 mutation determines sex-specific cardiac metabolism and identifies two novel cardiac metabolic regulators.

AIMS: Mutation of the PRDM16 gene causes human dilated and non-compaction cardiomyopathy. The PRDM16 protein is a transcriptional regulator that affects cardiac development via Tbx5 and Hand1, thus regulating myocardial structure. The biallelic inactivation of Prdm16 induces severe cardiac dysfunction with post-natal lethality and hypertrophy in mice. The early pathological events that occur upon Prdm16 inactivation have not been explored. METHODS AND RESULTS: This study performed in-depth pathophysiological and molecular analyses of male and female Prdm16csp1/wt mice that carry systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes through transcriptomics, proteomics, and metabolomics. Kinetic modelling of cardiac metabolism was performed in silico with CARDIOKIN. Prdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt cardiac tissue of both sexes showed reductions in metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and the tricarboxylic acid cycle. Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. An accumulation of triacylglycerides exclusively in male Prdm16csp1/wt hearts suggests a sex-specific metabolic adaptation. Metabolic modelling using CARDIOKIN identified a reduction in fatty acid utilization in males as well as lower glucose utilization in female Prdm16csp1/wt cardiac tissue. On the level of transcripts and protein expression, Prdm16csp1/wt hearts demonstrate an up-regulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre-B-cell leukaemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional up-regulation was detected for Prdm16 itself, probably through an autoregulatory mechanism. CONCLUSIONS: Monoallelic, global Prdm16 mutation diminishes cardiac performance in Prdm16csp1/wt mice. Metabolic alterations and transcriptional dysregulation in Prdm16csp1/wt affect cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype, indicating sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in the PRDM16 associated cardiac pathology.

CAR links hypoxia signaling to improved survival after myocardial infarction.

The coxsackievirus and adenovirus receptor (CAR) mediates homo- and heterotopic interactions between neighboring cardiomyocytes at the intercalated disc. CAR is upregulated in the hypoxic areas surrounding myocardial infarction (MI). To elucidate whether CAR contributes to hypoxia signaling and MI pathology, we used a gain- and loss-of-function approach in transfected HEK293 cells, H9c2 cardiomyocytes and CAR knockout mice. CAR overexpression increased RhoA activity, HIF-1α expression and cell death in response to chemical and physical hypoxia. In vivo, we subjected cardiomyocyte-specific CAR knockout (KO) and wild-type mice (WT) to coronary artery ligation. Survival was drastically improved in KO mice with largely preserved cardiac function as determined by echocardiography. Histological analysis revealed a less fibrotic, more compact lesion. Thirty days after MI, there was no compensatory hypertrophy or reduced cardiac output in hearts from CAR KO mice, in contrast to control mice with increased heart weight and reduced ejection fraction as signs of the underlying pathology. Based on these findings, we suggest CAR as a therapeutic target for the improved future treatment or prevention of myocardial infarction.

Mouse models of human multiple myeloma subgroups.

Multiple myeloma (MM), a tumor of germinal center (GC)-experienced plasma cells, comprises distinct genetic subgroups, such as the t(11;14)/CCND1 and the t(4;14)/MMSET subtype. We have generated genetically defined, subgroup-specific MM models by the GC B cell-specific coactivation of mouse Ccnd1 or MMSET with a constitutively active Ikk2 mutant, mimicking the secondary NF-κB activation frequently seen in human MM. Ccnd1/Ikk2ca and MMSET/Ikk2ca mice developed a pronounced, clonally restricted plasma cell outgrowth with age, accompanied by serum M spikes, bone marrow insufficiency, and bone lesions. The transgenic plasma cells could be propagated in vivo and showed distinct transcriptional profiles, resembling their human MM counterparts. Thus, we show that targeting the expression of genes involved in MM subgroup-specific chromosomal translocations into mouse GC B cells translates into distinct MM-like diseases that recapitulate key features of the human tumors, opening the way to a better understanding of the pathogenesis and therapeutic vulnerabilities of different MM subgroups.

Inhibition of the NLRP3/IL-1ß axis protects against sepsis-induced cardiomyopathy.

BACKGROUND: Septic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin-1ß (IL-1ß), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL-1ß in sepsis-induced cardiomyopathy and cardiac atrophy. METHODS: Male Nlrp3 knockout (KO) and wild-type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham-treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL-1ß effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real-time deformability cytometry (RT-DC) analysis were used to investigate functional and mechanical effects of IL-1ß on cardiomyocytes. RESULTS: Heart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 µL vs. 24.6 ± 8.7 µL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 µL vs. 29.9 ± 9.9 µL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight-tibia length ratio: WT CLP vs. WT sham: -26.6%, P < 0.05; KO CLP vs. KO sham: -3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL-1ß decreased contractility (cell shortening: IL-1ß: -15.4 ± 2.3%, P < 0.001 vs. vehicle, IL-1RA: -6.1 ± 3.3%, P < 0.05 vs. IL-1ß) and relaxation of adult rat ventricular cardiomyocytes (time-to-50% relengthening: IL-1ß: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL-1RA: 564 ± 247 ms, P < 0.001 vs. IL-1ß), which was attenuated by an IL-1 receptor antagonist (IL-1RA). RT-DC analysis indicated that IL-1ß reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF-κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL-1ß activated NF-κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL-1RA. CONCLUSIONS: IL-1ß causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL-1ß pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.

Age-related decline in murine heart and skeletal muscle performance is attenuated by reduced Ahnak1 expression.

BACKGROUND: Aging is associated with a progressive reduction in cellular function leading to poor health and loss of physical performance. Mitochondrial dysfunction is one of the hallmarks of aging; hence, interventions targeting mitochondrial dysfunction have the potential to provide preventive and therapeutic benefits to elderly individuals. Meta-analyses of age-related gene expression profiles showed that the expression of Ahnak1, a protein regulating several signal-transduction pathways including metabolic homeostasis, is increased with age, which is associated with low VO2MAX and poor muscle fitness. However, the role of Ahnak1 in the aging process remained unknown. Here, we investigated the age-related role of Ahnak1 in murine exercise capacity, mitochondrial function, and contractile function of cardiac and skeletal muscles. METHODS: We employed 15- to 16-month-old female and male Ahnak1-knockout (Ahnak1-KO) and wild-type (WT) mice and performed morphometric, biochemical, and bioenergetics assays to evaluate the effects of Ahnak1 on exercise capacity and mitochondrial morphology and function in cardiomyocytes and tibialis anterior (TA) muscle. A human left ventricular (LV) cardiomyocyte cell line (AC16) was used to investigate the direct role of Ahnak1 in cardiomyocytes. RESULTS: We found that the level of Ahnak1 protein is significantly up-regulated with age in the murine LV (1.9-fold) and TA (1.8-fold) tissues. The suppression of Ahnak1 was associated with improved exercise tolerance, as all aged adult Ahnak1-KO mice (100%) successfully completed the running programme, whereas approximately 31% male and 8% female WT mice could maintain the required running speed and distance. Transmission electron microscopic studies showed that LV and TA tissue specimens of aged adult Ahnak1-KO of both sexes have significantly more enlarged/elongated mitochondria and less small mitochondria compared with WT littermates (P < 0.01 and P < 0.001, respectively) at basal level. Further, we observed a shift in mitochondrial fission/fusion balance towards fusion in cardiomyocytes and TA muscle from aged adult Ahnak1-KO mice. The maximal and reserve respiratory capacities were significantly higher in cardiomyocytes from aged adult Ahnak1-KO mice compared with the WT counterparts (P < 0.05 and P < 0.01, respectively). Cardiomyocyte contractility and fatigue resistance of TA muscles were significantly increased in Ahnak1-KO mice of both sexes, compared with the WT groups. In vitro studies using AC16 cells have confirmed that the alteration of mitochondrial function is indeed a direct effect of Ahnak1. Finally, we presented Ahnak1 as a novel cardiac mitochondrial membrane-associated protein. CONCLUSIONS: Our data suggest that Ahnak1 is involved in age-related cardiac and skeletal muscle dysfunction and could therefore serve as a promising therapeutical target.

ONX 0914 Lacks Selectivity for the Cardiac Immunoproteasome in CoxsackievirusB3 Myocarditis of NMRI Mice and Promotes Virus-Mediated Tissue Damage.

: Inhibition of proteasome function by small molecules is highly efficacious in cancer treatment. Other than non-selective proteasome inhibitors, immunoproteasome-specific inhibitors allow for specific targeting of the proteasome in immune cells and the profound anti-inflammatory potential of such compounds revealed implications for inflammatory scenarios. For pathogen-triggered inflammation, however, the efficacy of immunoproteasome inhibitors is controversial. In this study, we investigated how ONX 0914, an immunoproteasome-selective inhibitor, influences CoxsackievirusB3 infection in NMRI mice, resulting in the development of acute and chronic myocarditis, which is accompanied by formation of the immunoproteasome in heart tissue. In groups in which ONX 0914 treatment was initiated once viral cytotoxicity had emerged in the heart, ONX 0914 had no anti-inflammatory effect in the acute or chronic stages. ONX 0914 treatment initiated prior to infection, however, increased viral cytotoxicity in cardiomyocytes, promoting infiltration of myeloid immune cells into the heart. At this stage, ONX 0914 completely inhibited the ß5 subunit of the standard cardiac proteasome and less efficiently blocked its immunoproteasome counterpart LMP7. In conclusion, ONX 0914 unselectively perturbs cardiac proteasome function in viral myocarditis of NMRI mice, reduces the capacity of the host to control the viral burden and promotes cardiac inflammation.

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.

Silencing the CSF-1 Axis Using Nanoparticle Encapsulated siRNA Mitigates Viral and Autoimmune Myocarditis.

Myocarditis is an inflammatory disease of the heart muscle most commonly caused by viral infection and often maintained by autoimmunity. Virus-induced tissue damage triggers chemokine production and, subsequently, immune cell infiltration with pro-inflammatory and pro-fibrotic cytokine production follows. In patients, the overall inflammatory burden determines the disease outcome. Following the aim to define specific molecules that drive both immunopathology and/or autoimmunity in inflammatory heart disease, here we report on increased expression of colony stimulating factor 1 (CSF-1) in patients with myocarditis. CSF-1 controls monocytes originating from hematopoietic stem cells and subsequent progenitor stages. Both, monocytes and macrophages are centrally involved in mediating tissue damage and fibrotic scarring in the heart. CSF-1 influences monocytes via engagement of CSF-1 receptor, and it is also produced by cells of the mononuclear phagocyte system themselves. Based on this, we sought to modulate the virus-triggered inflammatory response in an experimental model of Coxsackievirus B3-induced myocarditis by silencing the CSF-1 axis in myeloid cells using nanoparticle-encapsulated siRNA. siCSF-1 inverted virus-mediated immunopathology as reflected by lower troponin T levels, a reduction of accumulating myeloid cells in heart tissue and improved cardiac function. Importantly, pathogen control was maintained and the virus was efficiently cleared from heart tissue. Since viral heart disease triggers heart-directed autoimmunity, in a second approach we investigated the influence of CSF-1 upon manifestation of heart tissue inflammation during experimental autoimmune myocarditis (EAM). EAM was induced in Balb/c mice by immunization with a myocarditogenic myosin-heavy chain-derived peptide dissolved in complete Freund's adjuvant. siCSF-1 treatment initiated upon established disease inhibited monocyte infiltration into heart tissue and this suppressed cardiac injury as reflected by diminished cardiac fibrosis and improved cardiac function at later states. Mechanistically, we found that suppression of CSF-1 production arrested both differentiation and maturation of monocytes and their precursors in the bone marrow. In conclusion, during viral and autoimmune myocarditis silencing of the myeloid CSF-1 axis by nanoparticle-encapsulated siRNA is beneficial for preventing inflammatory tissue damage in the heart and preserving cardiac function without compromising innate immunity's critical defense mechanisms.

Desmoglein 2, but not desmocollin 2, protects intestinal epithelia from injury.

Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell-cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2ΔIEC; DSC2ΔIEC). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2ΔIEC, but not DSC2ΔIEC mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2ΔIEC mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2ΔIEC animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2ΔIEC mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2ΔIEC mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.

Regulation of body weight and energy homeostasis by neuronal cell adhesion molecule 1.

Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.

Muscle RING-finger 2 and 3 maintain striated-muscle structure and function.

BACKGROUND: The Muscle-specific RING-finger (MuRF) protein family of E3 ubiquitin ligases is important for maintenance of muscular structure and function. MuRF proteins mediate adaptation of striated muscles to stress. MuRF2 and MuRF3 bind to microtubules and are implicated in sarcomere formation with noticeable functional redundancy. However, if this redundancy is important for muscle function in vivo is unknown. Our objective was to investigate cooperative function of MuRF2 and MuRF3 in the skeletal muscle and the heart in vivo. METHODS: MuRF2 and MuRF3 double knockout mice (DKO) were generated and phenotypically characterized. Skeletal muscle and the heart were investigated by morphological measurements, histological analyses, electron microscopy, immunoblotting, and real-time PCR. Isolated muscles were subjected to in vitro force measurements. Cardiac function was determined by echocardiography and working heart preparations. Function of cardiomyocytes was measured in vitro. Cell culture experiments and mass-spectrometry were used for mechanistic analyses. RESULTS: DKO mice showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO soleus and extensor digitorum longus. Additionally, a fibre type shift towards slow/type I fibres occurred in DKO soleus and extensor digitorum longus. MuRF2 and MuRF3-deficient hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the myosin heavy chain isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in DKO hearts. CONCLUSIONS: The redundant function of MuRF2 and MuRF3 is important for maintenance of skeletal muscle and cardiac structure and function in vivo.

CD74-Downregulation of Placental Macrophage-Trophoblastic Interactions in Preeclampsia.

RATIONALE: We hypothesized that cluster of differentiation 74 (CD74) downregulation on placental macrophages, leading to altered macrophage-trophoblast interaction, is involved in preeclampsia. OBJECTIVE: Preeclamptic pregnancies feature hypertension, proteinuria, and placental anomalies. Feto-placental macrophages regulate villous trophoblast differentiation during placental development. Disturbance of this well-balanced regulation can lead to pathological pregnancies. METHODS AND RESULTS: We performed whole-genome expression analysis of placental tissue. CD74 was one of the most downregulated genes in placentas from preeclamptic women. By reverse transcriptase-polymerase chain reaction, we confirmed this finding in early-onset (<34 gestational week, n=26) and late-onset (≥34 gestational week, n=24) samples from preeclamptic women, compared with healthy pregnant controls (n=28). CD74 protein levels were analyzed by Western blot and flow cytometry. We identified placental macrophages to express CD74 by immunofluorescence, flow cytometry, and RT-PCR. CD74-positive macrophages were significantly reduced in preeclamptic placentas compared with controls. CD74-silenced macrophages showed that the adhesion molecules ALCAM, ICAM4, and Syndecan-2, as well as macrophage adhesion to trophoblasts were diminished. Naive and activated macrophages lacking CD74 showed a shift toward a proinflammatory signature with an increased secretion of tumor necrosis factor-α, chemokine (C-C motif) ligand 5, and monocyte chemotactic protein-1, when cocultured with trophoblasts compared with control macrophages. Trophoblasts stimulated by these factors express more CYP2J2, sFlt1, TNFα, and IL-8. CD74-knockout mice showed disturbed placental morphology, reduced junctional zone, smaller placentas, and impaired spiral artery remodeling with fetal growth restriction. CONCLUSIONS: CD74 downregulation in placental macrophages is present in preeclampsia. CD74 downregulation leads to altered macrophage activation toward a proinflammatory signature and a disturbed crosstalk with trophoblasts.

Inhibition of chymotryptic-like standard proteasome activity exacerbates doxorubicin-induced cytotoxicity in primary cardiomyocytes.

The anthracycline doxorubicin (DOX) is a potent anticancer agent for multiple myeloma (MM). A major limitation of this drug is the induction of death in cardiomyocytes leading to heart failure. Here we report on the role of the ubiquitin-proteasome system (UPS) as a critical surveillance pathway for preservation of cell vitality counteracting DOX treatment. Since in addition to DOX also suppression of proteasome activity is a rational therapeutic strategy for MM, we examined how small molecular compounds with clinically relevant proteasome subunit specificity affect DOX cytotoxicity. We found that during DOX-treatment, the activity of the ß5 standard proteasome subunit is crucial for limiting off-target cytotoxicity in primary cardiomyocytes. In contrast, we demonstrate that the ß5 equivalent LMP7 of the immunoproteasome represents a safe target for subunit-specific inhibitors in DOX-exposed cardiomyocytes. Neither inhibition of LMP7 in primary cardiomyocytes nor genetic ablation of LMP7 in heart tissue influenced the development of DOX cardiotoxicity. Our results indicate that as compared to compounds like carfilzomib, which target both the ß5 standard proteasome and the LMP7 immunoproteasome subunit, immunoproteasome-specific inhibitors with known anti-tumor capacity for MM cells might be advantageous for reducing cardiomyocyte death, when a combination therapy with DOX is envisaged.

Bcl10 mediates angiotensin II-induced cardiac damage and electrical remodeling.

Angiotensin (Ang) II is a potent mediator of both hypertension and cardiac damage; however, the mechanisms by which this occur remain unclear. B-cell lymphoma/leukemia 10 (Bcl10) is a member of the CBM signalosome, which links Ang II and nuclear factor-κB signaling. We hypothesized that Bcl10 is pivotal in the pathogenesis of Ang II-induced cardiac damage. Ang II infusion in mice lacking Bcl10 resulted in reduced cardiac fibrosis, less cellular infiltration, and improved arrhythmogenic electric remodeling, despite a similar degree of hypertension or cardiac hypertrophy. Adoptive transfer of bone marrow (BM), whereby Bcl10 knockout or wildtype BM was transferred to their opposite genotype recipients, revealed the dual importance of Bcl10 within both cardiac and immune cells. Loss of Bcl10 in cardiac cells resulted in reduced expression of genes important for the adhesion and recruitment of immune cells. In vitro experiments demonstrated that adhesion of monocytes to Ang II-treated endothelial cells also required Bcl10. Additionally, Bcl10 deficiency in macrophages reduced their intrinsic migratory ability. To address the role of BM-derived fibroblasts in the formation of cardiac fibrosis, we explored whether Bcl10 is also important for the infiltration of BM-derived (myo)fibroblasts into the heart. The transfer of green fluorescent protein positive wildtype BM into Bcl10 knockout recipient mice revealed a reduced number of noncardiac (myo)fibroblasts compared with those wildtype recipients. Our results demonstrate the significant role of Bcl10 in multiple cell types important for the generation of Ang II-induced cardiac damage and electric remodeling and may provide a new avenue for therapeutic intervention.

TAT-apoptosis repressor with caspase recruitment domain protein transduction rescues mice from fulminant liver failure.

UNLABELLED: Acute liver failure (ALF) is associated with massive hepatocyte cell death and high mortality rates. Therapeutic approaches targeting hepatocyte injury in ALF are hampered by the activation of distinct stimulus-dependent pathways, mechanism of cell death, and a limited therapeutic window. The apoptosis repressor with caspase recruitment domain (ARC) is a recently discovered death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. Here, we investigated the in vivo effects of ARC fused with the transduction domain of human immunodeficiency virus 1 (HIV-1) (TAT-ARC) on Fas- and tumor necrosis factor (TNF)-mediated murine models of fulminant liver failure. Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by Fas-agonistic antibody (Jo2), concanavalin A (ConA), or D-galactosamine/lipopolysaccharide (GalN/LPS) administration. Importantly, survival of mice was even preserved when TAT-ARC therapy was initiated in a delayed manner after stimulation with Jo2, ConA, or GalN/LPS. ARC blocked hepatocyte apoptosis by directly interacting with members of the death-inducing signaling complex. TNF-mediated liver damage was inhibited by two independent mechanisms: inhibition of jun kinase (JNK)-mediated TNF-α expression and prevention of hepatocyte apoptosis by inhibition of both death receptor and mitochondrial death signaling. We identified JNK as a novel target of ARC. ARC's caspase recruitment domain (CARD) directly interacts with JNK1 and JNK2, which correlates with decreased JNK activation and JNK-dependent TNF-α production. CONCLUSION: This work suggests that ARC confers hepatoprotection upstream and at the hepatocyte level. The efficacy of TAT-ARC protein transduction in multiple murine models of ALF demonstrates its therapeutic potential for reversing liver failure.

Mutant desmocollin-2 causes arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically heterogeneous heart-muscle disorder characterized by progressive fibrofatty replacement of right ventricular myocardium and an increased risk of sudden cardiac death. Mutations in desmosomal proteins that cause ARVC have been previously described; therefore, we investigated 88 unrelated patients with the disorder for mutations in human desmosomal cadherin desmocollin-2 (DSC2). We identified a heterozygous splice-acceptor-site mutation in intron 5 (c.631-2A-->G) of the DSC2 gene, which led to the use of a cryptic splice-acceptor site and the creation of a downstream premature termination codon. Quantitative analysis of cardiac DSC2 expression in patient specimens revealed a marked reduction in the abundance of the mutant transcript. Morpholino knockdown in zebrafish embryos revealed a requirement for dsc2 in the establishment of the normal myocardial structure and function, with reduced desmosomal plaque area, loss of the desmosome extracellular electron-dense midlines, and associated myocardial contractility defects. These data identify DSC2 mutations as a cause of ARVC in humans and demonstrate that physiologic levels of DSC2 are crucial for normal cardiac desmosome formation, early cardiac morphogenesis, and cardiac function.

Identification of a novel frameshift mutation in the giant muscle filament titin in a large Australian family with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is an etiologically heterogeneous cardiac disease characterized by left ventricular dilation and systolic dysfunction. Approximately 25-30% of DCM patients show a family history of mainly autosomal dominant inheritance. We and others have previously demonstrated that mutations in the giant muscle filament titin (TTN) can cause DCM. However, the prevalence of titin mutations in familial DCM is unknown. In this paper, we report a novel heterozygous 1-bp deletion mutation (c.62890delG) in TTN that cosegregates with DCM in a large Australian pedigree (A3). The TTN deletion mutation c.62890delG causes a frameshift, thereby generating a truncated A-band titin due to a premature stop codon (p.E20963KfsX10) and the addition of ten novel amino acid residues. The clinical phenotype of DCM in kindred A3 demonstrates incomplete penetrance and variable expressivity. Finally, protein analysis of a skeletal muscle biopsy sample from an affected member did not reveal the predicted truncated titin isoform although the aberrant mRNA was present, suggesting posttranslational modification and degradation of the truncated protein. The identification of a novel disease-causing mutation in the giant titin gene in a third large family with DCM indicates that mutations in titin may account for a significant portion of the genetic etiology in familial DCM.

Cardiac troponin C-L29Q, related to hypertrophic cardiomyopathy, hinders the transduction of the protein kinase A dependent phosphorylation signal from cardiac troponin I to C.

We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium-binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt-Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine-glutamine exchange at position 29 in the nonfunctional calcium-binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N-terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107-112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A-dependent phosphorylation of cTnI at serine 22 and serine 23 in wild-type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+-free and Ca2+-saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+-dependent actomyosin subfragment 1 (actoS1)-ATPase activity and the sliding velocity of thin filaments. The maximum actoS1-ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A-dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1-ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A-dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.

Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with fibrofatty replacement of cardiac myocytes, ventricular tachyarrhythmias and sudden cardiac death. In 32 of 120 unrelated individuals with ARVC, we identified heterozygous mutations in PKP2, which encodes plakophilin-2, an essential armadillo-repeat protein of the cardiac desmosome. In two kindreds with ARVC, disease was incompletely penetrant in most carriers of PKP2 mutations.