Patients with primary (AL) cardiac amyloidosis suffer from progressive cardiomyopathy with a median survival of less than 8 months and a 5-year survival of <10%. Contributing to this poor prognosis is the fact that these patients generally do not tolerate standard heart failure therapies. The molecular mechanisms underlying this deadly form of heart disease remain unclear. Although interstitial amyloid fibril deposition of Ig light chain proteins is a major cause of cardiac dysfunction in AL cardiac amyloidosis, we have previously shown that amyloid precursor proteins directly impair cardiac function at the cellular and isolated organ levels, independent of fibril formation. In this study, we report that amyloidogenic light chain (AL-LC) proteins provoke oxidative stress, cellular dysfunction, and apoptosis in isolated adult cardiomyocytes through activation of p38 mitogen-activated protein kinase (MAPK). AL-LC–induced p38 activation was found to be independent of the upstream MAPK kinase, MKK3/6, and instead depends upon transforming growth factor-β-activated protein kinase-1 binding protein-1 (TAB1)-mediated p38α MAPK autophosphorylation. Treatment of cardiomyocytes with SB203580, a selective p38 MAPK inhibitor, significantly attenuated AL-LC–induced oxidative stress, cellular dysfunction, and apoptosis. Our data provide a unique mechanistic insight into the pathogenesis of AL-LC cardiac toxicity and suggest that TAB1-mediated p38α MAPK autophosphorylation may serve as an important event leading to cardiac dysfunction and subsequent heart failure.

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CARD9 is dispensable for NF-κB activation induced by Dectin-1 ligands in mice. However, Dectin-1–induced H-Ras activation is mediated by a complex with CARD9, which leads to ERK activation for host innate immune responses to Candida albicans infection.

Background The arrangement of myofibers in the heart is highly complex and must be replicated by injected cells to produce functional myocardium. A novel approach to characterize the microstructural response of the myocardium to ischemia and cell therapy, using serial diffusion tensor MRI (DTI) tractography of the heart in vivo, is presented. Methods and Results Validation of the approach was performed in normal (n=6) and infarcted mice (n=6) as well as healthy human volunteers. Mice (n=12) were then injected with bone marrow mononuclear cells (BMMCs) 3 weeks after coronary ligation. In half the mice the donor and recipient strains were identical and in half the strains were different. A positive response to cell injection was defined by a decrease in mean diffusivity, an increase in fractional anisotropy, and the appearance of new myofiber tracts with the correct orientation. A positive response to BMMC injection was seen in one mouse. The response of the majority of mice to BMMC injection was neutral (9/12) or negative (2/12). The in vivo tractography findings were confirmed with histology. Conclusions DTI-tractography was able to directly resolve the ability of injected cells to generate new myofiber tracts and provided a fundamental readout of their regenerative capacity. A highly novel and translatable approach to assess the efficacy of cell therapy in the heart is thus presented.

Background-Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, is a multifunctional cytokine known to regulate cellular functions in contexts of injury and disease through its receptor, fibroblast growth factor-inducible molecule 14 (Fn14). Although many of the processes and downstream signals regulated by the TWEAK/Fn14 pathway have been implicated in the development of cardiac dysfunction, the role of TWEAK in the cardiovascular system is completely unknown. Methods and Results-Herein, we demonstrate that mouse and human cardiomyocytes express the TWEAK receptor Fn14. Furthermore, we determine that elevated circulating levels of TWEAK, induced via transgenic or adenoviralmediated gene expression in mice, result in dilated cardiomyopathy with subsequent severe cardiac dysfunction. This phenotype was mediated exclusively by the Fn14 receptor, independent of tumor necrosis factor-␣, and was associated with cardiomyocyte elongation and cardiac fibrosis but not cardiomyocyte apoptosis. Moreover, we find that circulating TWEAK levels were differentially upregulated in patients with idiopathic dilated cardiomyopathy compared with other forms of heart disease and normal control subjects. Conclusions-Our data suggest that TWEAK/Fn14 may be important in regulating myocardial structural remodeling and function and may play a role in the pathogenesis of dilated cardiomyopathy. Key Words: cardiomyocytes Ⅲ heart failure Ⅲ hypertrophy Ⅲ Fn14 TWEAK receptor Ⅲ Tweak protein, mouse M embers of the tumor necrosis factor (TNF) superfamily of cytokines represent a diverse group of signaling molecules that function as essential mediators of human disease, including the pathogenesis of cardiovascular disease. 1 TNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily of ligands, 2 is first synthesized as a type II transmembrane homotrimer and functions primarily as a soluble cytokine with diverse biological roles including proinflammatory activity, angiogenesis, and the regulation of cell survival, proliferation, and death. 3 TWEAK mediates these effects through its receptor Fn14, 4 a tightly regulated and inducible receptor, and has been suggested to signal through a variety of downstream signaling cascades, including via the nuclear factor-B, mitogen-activated protein kinase, and AKT pathways. [5][6][7] Whereas the expression of TWEAK has been identified across a range of tissues, including primary inflammatory cells, endothelial cells, and neurons, as well as numerous primary tumors and tumor cell lines, 3,8 Fn14 is expressed at relatively low levels in normal tissue. Importantly, Fn14 expression is highly upregulated in contexts of tissue injury and regeneration and chronic inflammatory disease, supporting a role for this pathway in physiological and pathological tissue remodeling. 3 Clinical Perspective p 2068Although many of the processes and downstream signals regulated by TWEAK/Fn14 have been implicated in the Received November 20, 2008; accepted...

Systemic AL amyloidosis results from the aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) usually produced by a plasma cell clone in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as amyloid fibrils can rapidly accumulate in tissues including the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. Chemotherapy is used to eradicate the cellular source of the amyloidogenic precursor. Currently, there are no therapies that target the process of LC aggregation, fibril formation, or organ damage. We developed transgenic mice expressing an amyloidogenic 6 LC using the cytomegalovirus ( IntroductionThe systemic amyloidoses are a diverse group of protein misfolding diseases in which proteins aggregate and form fibrillar deposits in tissues. Amyloidosis can be genetic in origin (familial amyloidosis, AF) or can occur in the setting of chronic inflammation or infection (amyloidosis because of deposition of the acute phase serum amyloid A protein, AA). However the most commonly diagnosed form, amyloid light chain (AL) amyloidosis, is because of deposition of an immunoglobulin light chain (LC) usually produced by clonal plasma cells in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as LC deposits may rapidly accumulate in organs such as the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. 1 Patients with AL amyloidosis are treated with chemotherapy to eradicate the plasma cell clone in the bone marrow that is the source of the amyloidogenic protein. Unfortunately, chemotherapeutics and even newer anti-plasma cell drugs with novel mechanisms of action can cause significant toxicity in AL amyloidosis patients. Although the pathophysiology of AL amyloidosis is still not completely understood, it is hoped that patient outcomes will be improved with the development of therapies that specifically target the process of protein aggregation, fibril formation, amyloid deposition, and organ damage.Although it is clear that the overexpression of a clonal amyloidogenic LC causes AL amyloidosis, it is not clear what structural features of amyloidogenic LC are responsible for misfolding and aggregation. Furthermore, although it is well-established that glycoaminoglycans 2 and serum amyloid P component 3 can interact with LC proteins, and are found in association with amyloid fibrils, the role of these accessory molecules in fibril formation in vivo is not well understood. The role of prefibrillar LCs in organ dysfunction remains a major question in the disease pathogenesis. Data from our group have demonstrated that amyloidogenic LC can be acutely toxic to target organs, inducing oxidative stress in cells and organ culture model systems. 4,5 Amyloidogenic LCs can be internalized into cells, regulating the expression of proteoglycans and possibly mediating interactions leading to the activation of stress and other signaling pathways. 6,7 Moreover, other investigators have demonstrat...

Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies.

We characterized the mutational landscape of human skin cutaneous melanoma (SKCM) using data obtained from The Cancer Genome Atlas (TCGA) project. We analyzed next-generation sequencing data of somatic copy number alterations and somatic mutations in 303 metastatic melanomas. We were able to confirm preeminent drivers of melanoma as well as identify new melanoma genes. The TCGA SKCM study confirmed a dominance of somatic BRAF mutations in 50% of patients. The mutational burden of melanoma patients is an order of magnitude higher than of other TCGA cohorts. A multi-step filter enriched somatic mutations while accounting for recurrence, conservation, and basal rate. Thus, this filter can serve as a paradigm for analysis of genome-wide next-generation sequencing data of large cohorts with a high mutational burden. Analysis of TCGA melanoma data using such a multi-step filter discovered novel and statistically significant potential melanoma driver genes. In the context of the Pan-Cancer study we report a detailed analysis of the mutational landscape of BRAF and other drivers across cancer tissues. Integrated analysis of somatic mutations, somatic copy number alterations, low pass copy numbers, and gene expression of the melanogenesis pathway shows coordination of proliferative events by Gs-protein and cyclin signaling at a systems level.