The rare mediastinal lipoma: a postmortem case report.

Mediastinal lipomas are extremely rare, so there are few reported cases. We report a postmortem case of this rare intrathoracic lipoma in a 79-year old female cadaver. The gross features of the tumor and histopathological findings confirmed the diagnosis of a massive simple benign lipoma. To our knowledge, this is the first case to be reported at such an advanced age and the second postmortem case found during comprehensive review of literature.

The role of medical museums in contemporary medical education.

From the early 19th century until the most recent two decades, open-space and satellite museums featuring anatomy and pathology collections (collectively referred to as "medical museums") had leading roles in medical education. However, many factors have caused these roles to diminish dramatically in recent years. Chief among these are the great advances in information technology and web-based learning that are currently at play in every level of medical training. Some medical schools have abandoned their museums while others have gradually given away their museums' contents to devote former museum space to new classrooms, lecture halls, and laboratories. These trends have accelerated as medical school enrollment has increased and as increasing interest in biological and biomedical research activities have caused medical schools to convert museum space into research facilities. A few medical schools, however, have considered the contents of their museums as irreplaceable resources for modern medicine and medical education and the space these occupy as great environments for independent and self-directed learning. Consequently, some medical schools have updated their medical museums and equipped them with new technologies. The Anatomical Museum of Leiden University Medical Center in The Netherlands and the Medical Museum of Kawasaki Medical School in Kurashiki, Okayama, Japan, are two examples of such upgraded museums. Student surveys at Leiden University have indicated that all students (100%) found audio-guided museum tours to be useful for learning and majorities of them found guided tours to be clinically relevant (87%). However, 69% of students felt that museum visits should be optional rather than compulsory within the medical training curriculum.

Cardiomyocyte degeneration with calpain deficiency reveals a critical role in protein homeostasis.

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 substrate-specific 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.

Cardiac-specific ablation of G-protein receptor kinase 2 redefines its roles in heart development and beta-adrenergic signaling.

G-protein receptor kinase 2 (GRK2) is 1 of 7 mammalian GRKs that phosphorylate ligand-bound 7-transmembrane receptors, causing receptor uncoupling from G proteins and potentially activating non-G-protein signaling pathways. GRK2 is unique among members of the GRK family in that its genetic ablation causes embryonic lethality. Cardiac abnormalities in GRK2 null embryos implicated GRK2 in cardiac development but prevented studies of the knockout phenotype in adult hearts. Here, we created GRK2-loxP-targeted mice and used Cre recombination to generate germline and cardiac-specific GRK2 knockouts. GRK2 deletion in the preimplantation embryo with EIIa-Cre (germline null) resulted in developmental retardation and embryonic lethality between embryonic day 10.5 (E10.5) and E11.5. At E9.5, cardiac myocyte specification and cardiac looping were normal, but ventricular development was delayed. Cardiomyocyte-specific ablation of GRK2 in the embryo with Nkx2.5-driven Cre (cardiac-specific GRK2 knockout) produced viable mice with normal heart structure, function, and cardiac gene expression. Cardiac-specific GRK2 knockout mice exhibited enhanced inotropic sensitivity to the beta-adrenergic receptor agonist isoproterenol, with impairment of normal inotropic and lusitropic tachyphylaxis, and exhibited accelerated development of catecholamine toxicity with chronic isoproterenol treatment. These findings show that cardiomyocyte autonomous GRK2 is not essential for myocardial development after cardiac specification, suggesting that embryonic developmental abnormalities may be attributable to extracardiac effects of GRK2 ablation. In the adult heart, cardiac GRK2 is a major factor regulating inotropic and lusitropic tachyphylaxis to beta-adrenergic agonist, which likely contributes to its protective effects in catecholamine cardiomyopathy.

Histidine-rich Ca binding protein: a regulator of sarcoplasmic reticulum calcium sequestration and cardiac function.

Defects in the pathways that regulate cardiac sarcoplasmic reticulum (SR) calcium (Ca) cycling represent prime targets for driving the deterioration of function and progression to heart failure. We hypothesized that the histidine-rich Ca binding protein (HRC) in the SR may be involved in SR Ca cycling and that alterations in HRC levels would result in abnormal cardiac Ca homeostasis. In order to test this hypothesis, we generated transgenic mice with cardiac overexpression (3-fold) of HRC. Increased cardiac HRC levels were associated with impaired SR Ca uptake rates (35%) and attenuated cardiomyocyte Ca transient decay (38%), without alterations in peak Ca transients or SR Ca load. The depressed SR Ca sequestration was associated with attenuated rate of Ca extrusion via Na-Ca exchange. Triadin protein expression levels and L-type Ca channel current density were increased, while the channel inactivation kinetics were not altered. Impaired SR Ca uptake and delayed Ca decline rates triggered hypertrophy and compromised the heart's responses to increased stress by either hemodynamic overload or the aging process. By 18 months of age, cardiac remodeling deteriorated to congestive heart failure in transgenic mice. Collectively, these data suggest that HRC may be an integral regulatory protein involved in cardiac muscle SR Ca uptake and Ca homeostasis.

The presence of Lys27 instead of Asn27 in human phospholamban promotes sarcoplasmic reticulum Ca2+-ATPase superinhibition and cardiac remodeling.

BACKGROUND: Phospholamban (PLN) is an inhibitor of the Ca2+ affinity of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2). The amino acid sequence of PLN is highly conserved, and although all species contain asparagine (Asn), human PLN is unique in containing lysine (Lys) at amino acid 27. METHODS AND RESULTS: Human PLN was introduced in the null background. Expression of human PLN, at similar levels to mouse wild-type PLN, resulted in significant decreases in the affinity of SERCA2 for Ca2+, attributed to unique spatial conformation of this PLN form and increases in its monomeric active unit compared with mouse PLN. The increased inhibition by human PLN was associated with attenuated cardiac contractility in the intact-animal, organ, and cardiomyocyte levels and with depressed calcium kinetics. These inhibitory effects could not be fully reversed even on maximal isoproterenol stimulation. There were no alterations in the expression levels of SERCA2, calsequestrin, ryanodine receptor, and FKBP12, although the sodium/calcium exchanger and the L-type Ca2+ channel expression levels were upregulated. The depressed function resulted in increased heart/body weight ratios and phosphorylation levels of Akt, p38, and Erk1/2. CONCLUSIONS: Human PLN may play a more inhibitory role than that of other species in Ca2+ cycling. Expression of human PLN in the mouse is compensated by alterations in Ca2+-handling proteins and cardiac remodeling in an effort to normalize cardiac contractility. Thus, the unique amino acid sequence of human PLN may be critical in maintaining a high cardiac reserve, which is of paramount importance in the regulation of human cardiac function.

Distinct pathways regulate proapoptotic Nix and BNip3 in cardiac stress.

Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 microM), angiotensin II (100 nM), or isoproterenol (10 microM). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKC alpha, but not PKC epsilon or dominant negative PKC alpha, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA- or PKC alpha-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G(q) overexpression model of hypertrophy. Luciferase activity increased in G alpha(q)-NixP hearts 3.2 +/- 0.4-fold and in TAC hearts 2.8 +/- 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxia-inducible, whereas Nix expression was induced by G alpha(q)-mediated hypertrophic stimuli. PKC alpha, a G(q) effector, transduced Nix transcriptional induction via Sp1.

Enhancement of cardiac function and suppression of heart failure progression by inhibition of protein phosphatase 1.

Abnormal calcium cycling, characteristic of experimental and human heart failure, is associated with impaired sarcoplasmic reticulum calcium uptake activity. This reflects decreases in the cAMP-pathway signaling and increases in type 1 phosphatase activity. The increased protein phosphatase 1 activity is partially due to dephosphorylation and inactivation of its inhibitor-1, promoting dephosphorylation of phospholamban and inhibition of the sarcoplasmic reticulum calcium-pump. Indeed, cardiac-specific expression of a constitutively active inhibitor-1 results in selective enhancement of phospholamban phosphorylation and augmented cardiac contractility at the cellular and intact animal levels. Furthermore, the beta-adrenergic response is enhanced in the transgenic hearts compared with wild types. On aortic constriction, the hypercontractile cardiac function is maintained, hypertrophy is attenuated and there is no decompensation in the transgenics compared with wild-type controls. Notably, acute adenoviral gene delivery of the active inhibitor-1, completely restores function and partially reverses remodeling, including normalization of the hyperactivated p38, in the setting of pre-existing heart failure. Thus, the inhibitor 1 of the type 1 phosphatase may represent an attractive new therapeutic target.

Physiological growth synergizes with pathological genes in experimental cardiomyopathy.

Hundreds of signaling molecules have been assigned critical roles in the pathogenesis of myocardial hypertrophy and heart failure based on cardiac phenotypes from alpha-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. Phenotype development was compared various times after neonatal (age 2 to 3 days) and adult (age 8 weeks) expression. Proapoptotic Nix caused ventricular dilation and severe contractile depression in neonates, but not adults. Myocardial apoptosis was minimal in adults, but was widespread in neonates, until it spontaneously resolved in adulthood. Unlike normal postnatal cardiac growth, concurrent left ventricular pressure overload hypertrophy did not synergize with Nix expression to cause cardiomyopathy or myocardial apoptosis. Prohypertrophic Galphaq likewise caused eccentric hypertrophy, systolic dysfunction, and pathological gene expression in neonates, but not adults. Thus, normal postnatal cardiac growth can be an essential cofactor in development of genetic cardiomyopathies, and may confound the interpretation of conventional alpha-MHC transgenic phenotypes.

Regulation of cardiac contractility by Rab4-modulated beta2-adrenergic receptor recycling.

Catecholaminergic activation of myocardial beta-adrenergic receptors (betaAR) is the principle mechanism regulating cardiac function. Agonists desensitize betaAR through G protein-coupled receptor kinase-mediated uncoupling and beta-arrestin-mediated internalization. Although inhibition of myocardial G protein-coupled receptor kinase-2 enhances cardiac function and reverses heart failure, pathophysiological effects of modulated betaAR internalization/recycling are unknown. We used mutation and transgenic expression of Rab4, which regulates vesicular transport of heptahelical receptors to plasma membranes, to interrogate in vivo betaAR trafficking and cardiac function. Expression of constitutively active Rab4 Q72L had no effects on cardiac structure or function, but dominant inhibitor Rab4 S27N impaired responsiveness to endogenous and exogenous catecholamines. To relate betaAR trafficking to diminished cardiac function, Rab4 mutant mice were crossbred with mice overexpressing human beta2AR. In unstimulated beta2AR overexpressors, beta2AR localized to heavier endosomes and translocated to lighter, caveolin-rich fractions after isoproterenol stimulation. Coexpression of beta2AR with activated Rab4 Q72L caused loss of receptors from heavier endosomes while retaining normal inotropy. In contrast, coexpression of beta2AR with inhibitory Rab4 S27N mimicked isoproterenol-induced receptor redistribution to caveolae, with diminished cardiac inotropy. Rab4 inhibition alone prevented resensitization after isoproterenol-induced in vivo adrenergic desensitization. Confocal and ultrastructural analyses revealed bizarre vesicular structures and abnormal accumulation of beta2AR in the sarcoplasm and subsarcollema of Rab4 S27N, but not Q72L, mice. These data provide evidence for constant bidirectional sarcollemal-vesicular betaAR trafficking in the in vivo heart and show that Rab4-mediated recycling of internalized betaAR is necessary for normal cardiac catecholamine responsiveness and resensitization after agonist exposure.

Increased particulate partitioning of PKC epsilon reverses susceptibility of phospholamban knockout hearts to ischemic injury.

Cytosolic Ca(2+) overload is a critical mediator of myocardial damage following cardiac ischemia-reperfusion. It has therefore been proposed that normalization of sarcoplasmic reticulum Ca(2+) cycling through inhibition or ablation of the Ca(2+) ATP-ase inhibitor phospholamban (PLN), which shows promise as a treatment for heart failure, could be beneficial in ischemic heart disease. However, a recent study has shown that globally ischemic PLN-deficient hearts exhibit increased ischemic injury, with impaired contractile, ATP, and phosphocreatine recoveries, compared to wild-type hearts. Since protein kinase C (PKC) family members are widely recognized as mediators of both post-ischemic injury and ischemic preconditioning, we assessed PKC levels in PLN-deficient hearts. Compared to genetically normal hearts, PLN-deficient hearts exhibited diminished particulate partitioning of PKC, a known cardioprotective PKC isoform, without alterations in the levels of membrane-associated PKC delta nor PKC alpha. To determine if decreased particulate partitioning of cardioprotective PKC epsilon was a cause of increased ischemic injury in PLN-deficient hearts, PLN-deficient mice were mated with mice expressing a myocardial-specific PKC epsilon translocation activator peptide, pseudo-epsilon receptor for activated kinase C (psi epsilon RACK). In psi epsilon RACK/PLN knockout (KO) hearts, PKC epsilon translocation to membranous cellular structures was augmented and this was associated with a significant acceleration of post-ischemic contraction and relaxation rates, as well as reduction of creatine phosphokinase release, compared to PLN-deficient hearts. Importantly, post-ischemic functional recovery reached pre-ischemic hyperdynamic values in psi epsilon RACK/PLN KO hearts, indicating super-rescue by the combination of PLN ablation and psi epsilon RACK expression. These findings suggest that diminished PKC epsilon particulate partitioning in PLN-deficient hearts is associated with attenuated contractile recovery upon ischemia-reperfusion and that increased translocation of PKC to membranous cellular structures confers full cardioprotection.

Protein kinase Calpha negatively regulates systolic and diastolic function in pathological hypertrophy.

The protein kinase C (PKC) family is implicated in cardiac hypertrophy, contractile failure, and beta-adrenergic receptor (betaAR) dysfunction. Herein, we describe the effects of gain- and loss-of-PKCalpha function using transgenic expression of conventional PKC isoform translocation modifiers. In contrast to previously studied PKC isoforms, activation of PKCalpha failed to induce cardiac hypertrophy, but instead caused betaAR insensitivity and ventricular dysfunction. PKCalpha inhibition had opposite effects. Because PKCalpha is upregulated in human and experimental cardiac hypertrophy and failure, its effects were also assessed in the context of the Galphaq overexpression model (in which PKCalpha is transcriptionally upregulated). Normalization (inhibition) of PKCalpha activity in Galpha(q) hearts improved systolic and diastolic function, whereas further activation of PKCalpha caused a lethal restrictive cardiomyopathy with marked interstitial fibrosis. These results define pathological roles for PKCalpha as a negative regulator of ventricular systolic and diastolic function.

Ischemic protection and myofibrillar cardiomyopathy: dose-dependent effects of in vivo deltaPKC inhibition.

To delineate the in vivo cardiac functions requiring normal delta protein kinase C (PKC) activity, we pursued loss-of-function through transgenic expression of a deltaPKC-specific translocation inhibitor protein fragment, deltaV1, in mouse hearts. Initial results using the mouse alpha-myosin heavy chain (alphaMHC) promoter resulted in a lethal heart failure phenotype. Viable deltaV1 mice were therefore obtained using novel attenuated mutant alphaMHC promoters lacking one or the other thyroid response element (TRE-1 and -2). In transgenic mouse hearts, deltaV1 decorated cytoskeletal elements and inhibited ischemia-induced deltaPKC translocation. At high levels, deltaV1 expression was uniformly lethal, with depressed cardiac contractile function, increased expression of fetal cardiac genes, and formation of intracardiomyocyte protein aggregates. Ultrastructural and immunoconfocal analyses of these aggregates revealed focal cytoskeletal disruptions and localized concentrations of desmin and alphaB-crystallin. In individual cardiomyocytes, cytoskeletal abnormalities correlated with impaired contractile function. Whereas desmin and alphaB-crystallin protein were increased approximately 4-fold in deltaV1 hearts, combined overexpression of these proteins at these levels was not sufficient to cause any detectable cardiac pathology. At low levels, deltaV1 expression conferred striking resistance to postischemic dysfunction, with no measurable effects on basal cardiac structure, function, or gene expression. Intermediate expression of deltaV1 conferred modest basal contractile depression with less ischemic protection, associated with abnormal cardiac gene expression, and a histological picture of infrequent cardiomyocyte cytoskeletal deformities. These results validate an approach of deltaPKC inhibition to protect against myocardial ischemia, but indicate that there is a threshold level of deltaPKC activation that is necessary to maintain normal cardiomyocyte cytoskeletal integrity.