A series of 88 conventional follicular and Hü rthle cell thyroid tumors were analyzed for RAS mutations and PAX8-PPAR␥ rearrangements using molecular methods and for galectin-3 and HBME-1 expression by immunohistochemistry. A novel LightCycler technology-based method was developed to detect point mutations in codons 12/13 and 61 of the H-RAS, K-RAS, and N-RAS genes. Forty-nine percent of conventional follicular carcinomas had RAS mutations, 36% had PAX8-PPAR␥ rearrangement, and only one (3%) had both. In follicular adenomas, 48% had RAS mutations, 4% had PAX8-PPAR␥ rearrangement, and 48% had neither. Follicular carcinomas with PAX8-PPAR␥ typically showed immunoreactivity for galectin-3 but not for HBME-1, tended to present at a younger patient age and be smaller size, and were almost always overtly invasive. In contrast, follicular carcinomas with RAS mutations most often displayed an HBME-1-positive/galectin-3-negative immunophenotype and were either minimally or overtly invasive. Hü rthle cell tumors infrequently had PAX8-PPAR␥ rearrangement or RAS mutations. These results suggest that conventional follicular thyroid carcinomas develop through at least two distinct and virtually nonoverlapping molecular pathways initiated by either RAS point mutation or PAX8-PPAR␥ rearrangement.

dilated cardiomyopathy ͉ heart failure ͉ calcium cycling ͉ mutation ͉ phosphorylation

Loss of cardiomyocytes through programmed cell death is a key event in the development of heart failure, but the inciting molecular mechanisms are largely unknown. We used microarray analysis to identify a genetic program for myocardial apoptosis in Gq-mediated and pressure-overload cardiac hypertrophy. A critical component of this apoptotic program was Nix/Bnip3L. Nix localized to mitochondria and caused release of cytochrome c, activation of caspase-3 and apoptotic cell death, when expressed in HEK293 fibroblasts. A previously undescribed truncated Nix isoform, termed sNix, was not targeted to mitochondria but heterodimerized with Nix and protected against Nix-mediated apoptosis. Forced in vivo myocardial expression of Nix resulted in apoptotic cardiomyopathy and rapid death. Conversely, sNix protected against apoptotic peripartum cardiomyopathy in G(alpha)q-overexpressors. Thus, Nix/Bnip3L is upregulated in myocardial hypertrophy, and is both necessary and sufficient for Gq-mediated apoptosis of cardiomyocytes and resulting hypertrophy decompensation.

In human disease and experimental animal models, depressed Ca2+ handling in failing cardiomyocytes is widely attributed to impaired sarcoplasmic reticulum (SR) function. In mice, disruption of the PLN gene encoding phospholamban (PLN) or expression of dominant-negative PLN mutants enhances SR and cardiac function, but effects of PLN mutations in humans are unknown. Here, a T116G point mutation, substituting a termination codon for Leu-39 (L39stop), was identified in two families with hereditary heart failure. The heterozygous individuals exhibited hypertrophy without diminished contractile performance. Strikingly, both individuals homozygous for L39stop developed dilated cardiomyopathy and heart failure, requiring cardiac transplantation at ages 16 and 27. An over 50% reduction in PLN mRNA and no detectable PLN protein were noted in one explanted heart. The expression of recombinant PLN-L39stop in human embryonic kidney (HEK) 293 cells and adult rat cardiomyocytes showed no PLN inhibition of SR Ca2+-ATPase and the virtual absence of stable PLN expression; where PLN was expressed, it was misrouted to the cytosol or plasma membrane. These findings describe a naturally-occurring loss-of-function human PLN mutation (PLN null). In contrast to reported benefits of PLN ablation in mouse heart failure, humans lacking PLN develop lethal dilated cardiomyopathy

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Abstract-Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substratespecific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation. (Circ Res.

Abstract-Caspase-1/interleukin-converting enzyme (ICE) is a cysteine protease traditionally considered to have importance as an inflammatory mediator, but not as an apoptotic effector. Because of the dual functions of this caspase, the pathophysiological impact of its reported upregulation in hypertrophy and heart failure is not known. Here, the consequences of increased myocardial expression of procaspase-1 were examined on the normal and ischemically injured heart. In unstressed mouse hearts with a 30-fold increase in procaspase-1 content, unprocessed procaspase-1 was well tolerated, without detectable pathology. Cardiomyocyte processing and activation of caspase-1 and caspase-3 occurred after administration of endotoxin or with transient myocardial ischemia. In post-ischemic hearts, procaspase-1 overexpression was associated with strikingly increased cardiac myocyte apoptosis in the peri-and noninfarct regions and with 50% larger myocardial infarctions. Tissue culture studies revealed that procaspase-1 processing/activation is stimulated by hypoxia, and that caspase-1 acts in synergy with hypoxia to stimulate caspase-3 mediated apoptosis without activating upstream caspases. These data demonstrate that the proapoptotic effects of caspase-1 can significantly impact the myocardial response to ischemia and suggest that conditions in which procaspase-1 in the heart is increased may predispose to apoptotic myocardial injury under conditions of physiological stress.

Abstract-Hundreds of signaling molecules have been assigned critical roles in the pathogenesis of myocardial hypertrophy and heart failure based on cardiac phenotypes from ␣-myosin heavy chain-directed overexpression mice. Because permanent ventricular transgene expression in this system begins during a period of rapid physiological neonatal growth, resulting phenotypes are the combined consequences of transgene effects and normal trophic influences. We used temporally-defined forced gene expression to investigate synergy between postnatal physiological cardiac growth and two functionally divergent cardiomyopathic genes.