Defective proteolytic systems in Mybpc3-targeted mice with cardiac hypertrophy.

Several lines of evidence suggest that alterations of the ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) may be involved in cardiac diseases. Little is known, however, in hypertrophic cardiomyopathy (HCM). This study studied these pathways in two mouse models of HCM that mainly differ by the presence or absence of truncated mutant proteins. Analyses were performed in homozygous Mybpc3-targeted knock-in (KI) mice, carrying a HCM mutation and exhibiting low levels of mutant cardiac myosin-binding protein C (cMyBP-C), and in Mybpc3-targeted knock-out (KO) mice expressing no cMyBP-C, thus serving as a model of pure cMyBP-C insufficiency. In the early postnatal development of cardiac hypertrophy, both models showed higher levels of ubiquitinated proteins and greater proteasomal activities. To specifically monitor the degradation capacity of the UPS with age, mice were crossed with transgenic mice that overexpress Ub(G76V)-GFP. Ub(G76V)-GFP protein levels were fourfold higher in 1-year-old KI, but not KO mice, suggesting a specific UPS impairment in mice expressing truncated cMyBP-C. Whereas protein levels of key ALP markers were higher, suggesting ALP activation in both mutant mice, their mRNA levels did not differ between the groups, underlying rather defective ALP-mediated degradation. Analysis of key proteins regulated in heart failure did not reveal specific alterations in KI and KO mice. Our data suggest (1) UPS activation in early postnatal development of cardiac hypertrophy, (2) specific UPS impairment in old KI mice carrying a HCM mutation, and (3) defective ALP as a common mechanism in genetically engineered mice with cardiac hypertrophy.

Plasminogen activator inhibitor type 1 up-regulation is associated with skeletal muscle atrophy and associated fibrosis.

Muscle wasting remains a feature of many diseases and is counteracted by anabolic supplementation or exercise. Persisting atrophy-inducing conditions can be complicated by skeletal muscle fibrosis, which leads to functional impairment. Identification of early mechanisms that initiate atrophy-induced fibrosis may reveal novel targets for therapy or diagnosis. Therefore, we investigated changes in the expression of genes involved in extracellular matrix homeostasis during glucocorticoid-induced atrophy of myotubes and compared them with insulin-like growth factor-1-induced hypertrophy. Obtained results were verified in rat gastrocnemius muscle that was exposed to microgravity by space flight for 2 weeks. Myostatin and atrogin-1 mRNA levels reflected the magnitude of atrophy. Despite differential induction of these negative muscle mass regulators, no major changes in matrix metalloproteinases-2, -9, and -14 mRNAs or their physiological inhibitors could be detected in either atrophy model. In contrast, transcript levels of plasminogen activator inhibitor type 1 (PAI-1) was dramatically increased in atrophic myotubes and microgravity-exposed rat gastrocnemius muscle, while plasminogen activators remained unaltered. In contrast to atrophy, no increase in PAI-1 mRNA levels could be detected in rat hindlimb that was electrically stimulated for 21 days. Furthermore, a strong increase in PAI-1 mRNA levels was identified in skeletal muscle of patients with neurogenic muscle atrophy. Our study suggests that increased PAI-1 expression in atrophic skeletal muscle may lead to muscle fibrosis by reducing plasmin generation.

Cardiac myosin-binding protein C is required for complete relaxation in intact myocytes.

The role of cardiac myosin-binding protein C (cMyBP-C) in cardiac contraction is still not fully resolved. Experimental ablation of cMyBP-C by various means resulted in inconsistent changes in Ca2+ sensitivity and increased velocity of force of skinned preparations. To evaluate how these effects are integrated in an intact, living myocyte context, we investigated consequences of cMyBP-C ablation in ventricular myocytes and left atria from cMyBP-C knock-out (KO) mice compared with wild-type (WT). At 6 weeks, KO myocytes exhibited mild hypertrophy that became more pronounced by 30 weeks. Isolated cells from KO exhibited markedly lower diastolic sarcomere length (SL) without change in diastolic Ca2+. The lower SL in KO was partly abolished by the actin-myosin ATPase inhibitors 2,3-butanedione monoxime or blebbistatin, indicating residual actin-myosin interaction in diastole. The relationship between cytosolic Ca2+ and SL showed that KO cells started to contract at lower Ca2+ without reaching a higher maximum, yielding a smaller area of the phase-plane diagram. Both sarcomere shortening and Ca2+ transient were prolonged in KO. Isolated KO left atria exhibited a marked increase in sensitivity to external Ca2+ and, in contrast to WT, continued to develop twitch force at low micromolar Ca2+. Taken together, the main consequence of cMyBP-C ablation was a defect in diastolic relaxation and a smaller dynamic range of cell shortening, both of which likely result from the increased myofilament Ca2+ sensitivity. Our findings indicate that cMyBP-C functions as a restraint on myosin-actin interaction at low Ca2+ and short SL to allow complete relaxation during diastole.

Expression of heat shock proteins in tissues from young pigs exposed to transport stress.

The objective of the present study was to investigate the role of heat shock proteins (Hsps) as potential stress response marker in several organs of transported pigs. Constitutive (Hsp90, Hsp70 and Hsp27) and inducible (Hsp72 and Hsp86) Hsps expressed in skeletal muscle, heart, liver and kidney of transported young pigs were investigated. The study comprised 13 German Landrace line pigs (mean weight, approx. 35 +/- 1 kg). Eight animals (n = 8) were transported for a period of 6 h, whereas the control group (n = 5) was kept under normal housing conditions. Identification of Hsps was performed by immunoblot using porcine specific antibodies. Although the 5 Hsps were regularly detected in all porcine tissues, no clear up-regulation could be observed due to transportation. Densitometrical analysis of the immunoblots revealed an unexpected result. Every tissue had a significant reduction of at least 2 Hsp members: Kidney (Hsp90 and Hsp70), M. longissimus dorsi (Hsp90 and Hsp72) and M. gluteus maximus superficialis (Hsp90 and Hsp86), liver (Hsp90, Hsp86 and Hsp27). The heart was most affected, all Hsps were significantly reduced by 26% to 41% after 6 h of transportation. The regular reduction of the large Hsp90 and Hsp86 in nearly all tissues examined point to a new and critical role these Hsps might have in counteracting short-term stress reactions. The fast reduction of Hsps in fatal organs such as heart and kidney may have a relation to organ failure. Hsp90 level may therefore serve as a potential marker for the stress pigs are suffering during transportation.

Inhibition of restenosis development after mechanical injury: a new field of application for malononitrilamides?

OBJECTIVE: To investigate the efficacy of the malononitrilamide FK778 to prevent vascular smooth muscle cell (SMC) migration/proliferation, and vascular fibrosis, the key events in restenosis development using in vivo and in vitro studies. BACKGROUND: Since the high rate of restenosis after percutaneous transluminal coronary angioplasty limited its long-term success, the implementation of locally delivered antiproliferative/immunosuppressive agents became advantageous. METHODS: Rats underwent balloon denudation of the abdominal aorta and received sirolimus, tacrolimus, or FK778 for 28 days in varying doses. Aortas were harvested for histologic evaluation, profibrotic gene expression, and organ chamber studies. Antifibrotic, antiproliferative and antimigratory effects of the immunosuppressants were further evaluated in vitro. RESULTS: Histology of untreated animals revealed marked intimal hyperplasia with moderate luminal obliteration. Neointima formation was dose-dependently attenuated by all three agents with FK778 and sirolimus being most efficacious. Organ chamber relaxation studies showed a leftward shift of the nitroglycerin and the acetylcholine dose-responses in all treatment groups, indicating diminished endothelial dysfunction. In vivo, only FK778 treatment revealed a significant downregulation of the TGF-beta/vasorin system which could be explained by upregulation of the TGF-beta-inhibitory mediator SMAD7. In vitro, FK778 showed most potent antiproliferative and antimigratory effects on SMC compared with sirolimus and tacrolimus. Only the antiproliferative effect of FK778 was due to pyrimidine synthesis blockade and could be reversed by uridine supplementation. CONCLUSIONS: The malononitrilamide FK778 proved highly efficacious against restenosis development by targeting two major components of intimal hyperplasia: SMC proliferation/migration and vascular fibrosis. Thus, the introduction of malononitrilamide-loaded stents may be a promising effort for future strategies.

Impact of AT2 receptor deficiency on postnatal cardiovascular development.

BACKGROUND: The angiotensin II receptor subtype 2 (AT2 receptor) is ubiquitously and highly expressed in early postnatal life. However, its role in postnatal cardiac development remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: Hearts from 1, 7, 14 and 56 days old wild-type (WT) and AT2 receptor-deficient (KO) mice were extracted for histomorphometrical analysis as well as analysis of cardiac signaling and gene expression. Furthermore, heart and body weights of examined animals were recorded and echocardiographic analysis of cardiac function as well as telemetric blood pressure measurements were performed. Moreover, gene expression, sarcomere shortening and calcium transients were examined in ventricular cardiomyocytes isolated from both genotypes. KO mice exhibited an accelerated body weight gain and a reduced heart to body weight ratio as compared to WT mice in the postnatal period. However, in adult KO mice the heart to body weight ratio was significantly increased most likely due to elevated systemic blood pressure. At postnatal day 7 ventricular capillarization index and the density of α-smooth muscle cell actin-positive blood vessels were higher in KO mice as compared to WT mice but normalized during adolescence. Echocardiographic assessment of cardiac systolic function at postnatal day 7 revealed decreased contractility of KO hearts in response to beta-adrenergic stimulation. Moreover, cardiomyocytes from KO mice showed a decreased sarcomere shortening and an increased peak Ca(2+) transient in response to isoprenaline when stimulated concomitantly with angiotensin II. CONCLUSION: The AT2 receptor affects postnatal cardiac growth possibly via reducing body weight gain and systemic blood pressure. Moreover, it moderately attenuates postnatal vascularization of the heart and modulates the beta adrenergic response of the neonatal heart. These AT2 receptor-mediated effects may be implicated in the physiological maturation process of the heart.

Quantification of hormone-induced atrophy of large myotubes from C2C12 and L6 cells: atrophy-inducible and atrophy-resistant C2C12 myotubes.

Myofiber atrophy is the final outcome of muscle wasting induced by catabolic factors such as glucocorticoids and thyroid hormones. We set up an in vitro system to define the catabolic reaction based on myotube atrophy. Both mouse C(2)C(12) and rat L6 cells were used. C(2)C(12) myotube formation was improved by replacing horse serum with the serum substitute Ultroser G. A new method was developed to quantify size changes of large (0.5-1 mm) myotubes only, excluding remaining myoblasts and small myotubes. Dexamethasone reduced myotube size by 30% in L6 but not in C(2)C(12) myotubes. Expression of the glucocorticoid receptor was twofold higher in L6 myotubes than in C(2)C(12) myotubes. In both cell lines, 3,3',5-triiodo-l-thyronine (T(3)) did not induce a significant size reduction. Expression of the major T(3) receptor (T(3)Rbeta1) was higher in L6 myotubes. We investigated whether the changes in myotube size are related to changes in atrogin-1 expression, as this enzyme is thought to be a key factor in the initiation of muscle atrophy. Dexamethasone induced a twofold increase of atrogin-1 mRNA; again, only L6 myotubes were susceptible. Interestingly, atrogin-1 expression in Ultroser G-fused C(2)C(12) myotubes was lower than that in horse serum-fused myotubes. Furthermore, dexamethasone treatment increased atrogin-1 expression only in horse serum-fused myotubes but not in Ultroser G-fused myotubes. Ultroser G-induced fusion may result in atrophy-resistant C(2)C(12) myotubes. Therefore, C(2)C(12) myotubes offer an ideal system in which to study skeletal muscle atrophy because, depending on differentiation conditions, C(2)C(12) cells produce atrophy-inducible and atrophy-resistant myotubes.