Hyperphosphorylation of mouse cardiac titin contributes to transverse aortic constriction-induced diastolic dysfunction.

RATIONALE: Mechanisms underlying diastolic dysfunction need to be better understood. OBJECTIVE: To study the role of titin in diastolic dysfunction using a mouse model of experimental heart failure induced by transverse aortic constriction. METHODS AND RESULTS: Eight weeks after transverse aortic constriction surgery, mice were divided into heart failure (HF) and congestive heart failure (CHF) groups. Mechanical studies on skinned left ventricle myocardium measured total and titin-based and extracellular matrix-based passive stiffness. Total passive stiffness was increased in both HF and CHF mice, and this was attributable to increases in both extracellular matrix-based and titin-based passive stiffness, with titin being dominant. Protein expression and titin exon microarray analysis revealed increased expression of the more compliant N2BA isoform at the expense of the stiff N2B isoform in HF and CHF mice. These changes are predicted to lower titin-based stiffness. Because the stiffness of titin is also sensitive to titin phosphorylation by protein kinase A and protein kinase C, back phosphorylation and Western blot assays with novel phospho-specific antibodies were performed. HF and CHF mice showed hyperphosphorylation of protein kinase A sites and the proline glutamate valine lysine (PEVK) S26 protein kinase C sites, but hypophosphorylation of the PEVK S170 protein kinase C site. Protein phosphatase I abolished differences in phosphorylation levels and normalized titin-based passive stiffness levels between control and HF myocardium. CONCLUSION: Transverse aortic constriction-induced HF results in increased extracellular matrix-based and titin-based passive stiffness. Changes in titin splicing occur, which lower passive stiffness, but this effect is offset by hyperphosphorylation of residues in titin spring elements, particularly of PEVK S26. Thus, complex changes in titin occur that combined are a major factor in the increased passive myocardial stiffness in HF.

Reduced myofibrillar connectivity and increased Z-disk width in nebulin-deficient skeletal muscle.

A prominent feature of striated muscle is the regular lateral alignment of adjacent sarcomeres. An important intermyofibrillar linking protein is the intermediate filament protein desmin, and based on biochemical and structural studies in primary cultures of myocytes it has been proposed that desmin interacts with the sarcomeric protein nebulin. Here we tested whether nebulin is part of a novel biomechanical linker complex, by using a recently developed nebulin knockout (KO) mouse model and measuring Z-disk displacement in adjacent myofibrils of both extensor digitorum longus (EDL) and soleus muscle. Z-disk displacement increased as sarcomere length (SL) was increased and the increase was significantly larger in KO fibers than in wild-type (WT) fibers; results in 3-day-old and 10-day-old mice were similar. Immunoelectron microscopy revealed reduced levels of desmin in intermyofibrillar spaces adjacent to Z-disks in KO fibers compared with WT fibers. We also performed siRNA knockdown of nebulin and expressed modules within the Z-disk portion of nebulin (M160-M170) in quail myotubes and found that this prevented the mature Z-disk localization of desmin filaments. Combined, these data suggest a model in which desmin attaches to the Z-disk through an interaction with nebulin. Finally, because nebulin has been proposed to play a role in specifying Z-disk width, we also measured Z-disk width in nebulin KO mice. Results show that most Z-disks of KO mice were modestly increased in width (approximately 80 nm in soleus and approximately 40 nm in EDL fibers) whereas a small subset had severely increased widths (up to approximately 1 microm) and resembled nemaline rod bodies. In summary, structural studies on a nebulin KO mouse show that in the absence of nebulin, Z-disks are significantly wider and that myofibrils are misaligned. Thus the functional roles of nebulin extend beyond thin filament length regulation and include roles in maintaining physiological Z-disk widths and myofibrillar connectivity.

Titin is a target of matrix metalloproteinase-2: implications in myocardial ischemia/reperfusion injury.

BACKGROUND: Titin is the largest mammalian (≈3000 to 4000 kDa) and myofilament protein that acts as a molecular spring in the cardiac sarcomere and determines systolic and diastolic function. Loss of titin in ischemic hearts has been reported, but the mechanism of titin degradation is not well understood. Matrix metalloproteinase-2 (MMP-2) is localized to the cardiac sarcomere and, on activation in ischemia/reperfusion injury, proteolyzes specific myofilament proteins. Here we determine whether titin is an intracellular substrate for MMP-2 and if its degradation during ischemia/reperfusion contributes to cardiac contractile dysfunction. METHODS AND RESULTS: Immunohistochemistry and confocal microscopy in rat and human hearts showed discrete colocalization between MMP-2 and titin in the Z-disk region of titin and that MMP-2 is localized mainly to titin near the Z disk of the cardiac sarcomere. Both purified titin and titin in skinned cardiomyocytes were proteolyzed when incubated with MMP-2 in a concentration-dependent manner, and this was prevented by MMP inhibitors. Isolated rat hearts subjected to ischemia/reperfusion injury showed cleavage of titin in ventricular extracts by gel electrophoresis, which was confirmed by reduced titin immunostaining in tissue sections. Inhibition of MMP activity with ONO-4817 prevented ischemia/reperfusion-induced titin degradation and improved the recovery of myocardial contractile function. Titin degradation was also reduced in hearts from MMP-2 knockout mice subjected to ischemia/reperfusion in vivo compared with wild-type controls. CONCLUSION: MMP-2 localizes to titin at the Z-disk region of the cardiac sarcomere and contributes to titin degradation in myocardial ischemia/reperfusion injury.

PKC phosphorylation of titin's PEVK element: a novel and conserved pathway for modulating myocardial stiffness.

RATIONALE: Protein kinase C (PKC) regulates contractility of cardiac muscle cells by phosphorylating thin- and thick- filament-based proteins. Myocardial sarcomeres also contain a third myofilament, titin, and it is unknown whether titin can be phosphorylated by PKC and whether it affects passive tension. OBJECTIVE: The purpose of this study was to examine the effect of PKC on titin phosphorylation and titin-based passive tension. METHODS AND RESULTS: Phosphorylation assays with PKCalpha revealed that titin is phosphorylated in skinned myocardial tissues; this effect is exacerbated by pretreating with protein phosphatase 1. In vitro phosphorylation of recombinant protein representing titin's spring elements showed that PKCalpha targets the proline - glutamate - valine - lysine (PEVK) spring element. Furthermore, mass spectrometry in combination with site-directed mutagenesis identified 2 highly conserved sites in the PEVK region that are phosphorylated by PKCalpha (S11878 and S12022); when these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that PKCalpha significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKCalpha decreases the PEVK persistence length (from 1.20 nm to 0.55 nm), without altering the contour length, and using a serially-linked wormlike chain model we show that this increases titin-based passive force with a sarcomere length dependence that is similar to that measured in skinned myocardium after PKCalpha phosphorylation. CONCLUSIONS: PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.

Excision of titin's cardiac PEVK spring element abolishes PKCalpha-induced increases in myocardial stiffness.

Protein kinase C-alpha (PKCalpha) was recently reported to increase myocardial stiffness, an effect that was proposed to be due to phosphorylation of two highly conserved sites (S11878 and S12022) within the proline-glutamic acid-valine-lysine (PEVK) rich spring element of titin. To test this proposal we investigated the effect of PKCalpha on phosphorylation and passive stiffness in a mouse model lacking the titin exons that contain these two phosphorylation sites, the PEVK knockout (KO). We used skinned, gelsolin-extracted, left ventricular myocardium from wildtype and PEVK KO mice. Consistent with previous work we found that PKCalpha increased passive stiffness in the WT myocardium by 27+/-6%. Importantly, this effect was completely abolished in KO myocardium. In addition, increases in the elastic and viscous moduli at a wide range of frequencies (properties important in diastolic filling) following PKCalpha incubation (27+/-3% and 20+/-4%, respectively) were also ablated in the KO. Back phosphorylation assays showed that titin phosphorylation following incubation with PKCalpha was significantly reduced by 36+/-12% in skinned PEVK KO myocardial tissues. The remaining phosphorylation in the KO suggests that PKCalpha sites exist in the titin molecule outside the PEVK region; these sites are not involved in increasing passive stiffness. Our results firmly support that the PEVK region of cardiac titin is phosphorylated by PKCalpha and that this increases passive tension. Thus, the PEVK spring element is the critical site of PKCalpha's involvement in passive myocardial stiffness.

Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development.

We studied differential splicing of nebulin, a giant filamentous F-actin binding protein (M(r) approximately 700-800kDa) that is found in skeletal muscle. Nebulin spans the thin filament length, its C-terminus is anchored in the Z-disc, and its N-terminal region is located toward the thin filament pointed end. Various lines of evidence indicate that nebulin plays important roles in thin filament and Z-disc structure in skeletal muscle. In the present work we studied nebulin in a range of muscle types during postnatal development and performed transcript studies with a mouse nebulin exon microarray, developed by us, whose results were confirmed by RT-PCR. We also performed protein studies with high-resolution SDS-agarose gels and Western blots, and structural studies with electron microscopy. We found during postnatal development of the soleus muscle major changes in splicing in both the super-repeat region and the Z-disc region of nebulin; interestingly, these changes were absent in other muscle types. Three novel Z-disc exons, previously described in the mouse gene, were upregulated during postnatal development of soleus muscle and this was correlated with a significant increase in Z-disc width. These findings support the view that nebulin plays an important role in Z-disc width regulation. In summary, we discovered changes in both the super-repeat region and the Z-disc region of nebulin, that these changes are muscle-type specific, and that they correlate with differences in sarcomere structure.

Stress-induced dilated cardiomyopathy in a knock-in mouse model mimicking human titin-based disease.

Mutations in a variety of myofibrillar genes cause dilated cardiomyopathy (DCM) in humans, usually with dominant inheritance and incomplete penetrance. Here, we sought to clarify the functional effects of the previously identified DCM-causing TTN 2-bp insertion mutation (c.43628insAT) and generated a titin knock-in mouse model mimicking the c.43628insAT allele. Mutant embryos homozygous for the Ttn knock-in mutation developed defects in sarcomere formation and consequently died before E9.5. Heterozygous mice were viable and demonstrated normal cardiac morphology, function and muscle mechanics. mRNA and protein expression studies on heterozygous hearts demonstrated elevated wild-type titin mRNA under resting conditions, suggesting that up-regulation of the wild-type titin allele compensates for the unstable mutated titin under these conditions. When chronically exposed to angiotensin II or isoproterenol, heterozygous mice developed marked left ventricular dilatation (p<0.05) with impaired fractional shortening (p<0.001) and diffuse myocardial fibrosis (11.95+/-2.8% vs. 3.7+/-1.1%). Thus, this model mimics typical features of human dilated cardiomyopathy and may further our understanding of how titin mutations perturb cardiac function and remodel the heart.

Hypothalamic and hindbrain NPY, AGRP and NE increase consummatory feeding responses.

Feeding behavior is comprised of both appetitive and consummatory responses to food. Appetitive responses include the motivated acquisition of food. Consummatory responses, including swallowing, are those that move the food from the mouth to the stomach. Intraoral delivery of liquid food bypasses the requirement for appetitive responses and has been used to examine consummatory responses directly in intact rats. In the present study, we administered neuropeptide Y (NPY), agouti-related protein (AGRP) and norepinephrine (NE), into the paraventricular nucleus of the hypothalamus (PVN) or into the fourth cerebral ventricle to examine their effects on the consummatory component of feeding behavior in the rat. To measure consummatory responses, milk (40% lactose free cow's milk diluted with water) was infused intraorally through a chronic cheek fistula (1 ml/min), using an alternating 5 min on -1 min off schedule, until rejection occurred. We found that both hypothalamic and fourth ventricle injections of NPY, AGRP and NE significantly increased consumption of the intraorally-delivered milk. Our results indicate that the circuitry for modulation of consummatory ingestive responses includes NE, NPY and AGRP receptors operating in both hypothalamic and hindbrain sites.

Cancer-Osteoblast Interaction Reduces Sost Expression in Osteoblasts and Up-Regulates lncRNA MALAT1 in Prostate Cancer.

Dynamic interaction between prostate cancer and the bone microenvironment is a major contributor to metastasis of prostate cancer to bone. In this study, we utilized an in vitro co-culture model of PC3 prostate cancer cells and osteoblasts followed by microarray based gene expression profiling to identify previously unrecognized prostate cancer-bone microenvironment interactions. Factors secreted by PC3 cells resulted in the up-regulation of many genes in osteoblasts associated with bone metabolism and cancer metastasis, including Mmp13, Il-6 and Tgfb2, and down-regulation of Wnt inhibitor Sost. To determine whether altered Sost expression in the bone microenvironment has an effect on prostate cancer metastasis, we co-cultured PC3 cells with Sost knockout (Sost(KO)) osteoblasts and wildtype (WT) osteoblasts and identified several genes differentially regulated between PC3-Sost(KO) osteoblast co-cultures and PC3-WT osteoblast co-cultures. Co-culturing PC3 cells with WT osteoblasts up-regulated cancer-associated long noncoding RNA (lncRNA) MALAT1 in PC3 cells. MALAT1 expression was further enhanced when PC3 cells were co-cultured with Sost(KO) osteoblasts and treatment with recombinant Sost down-regulated MALAT1 expression in these cells. Our results suggest that reduced Sost expression in the tumor microenvironment may promote bone metastasis by up-regulating MALAT1 in prostate cancer.

SOST Inhibits Prostate Cancer Invasion.

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings.

Hindbrain catecholamine neurons mediate consummatory responses to glucoprivation.

Previous work using the retrogradely transported immunotoxin, saporin (SAP) conjugated to a monoclonal antibody against dopamine-beta-hydroxylase (DBH; DSAP), to selectively lesion norepinephrine (NE) and epinephrine (E) neurons projecting to the medial hypothalamus, demonstrated the essential role of these neurons for appetitive ingestive responses to glucoprivation. Here, we again utilized this lesion to assess the importance of these same neurons for the consummatory phase of glucoprivic feeding. To test consummatory responses, milk was infused intraorally through a chronic cheek fistula until rejected. Appetitive responses were tested in the same rats using pelleted food. Feeding responses to insulin-induced hypoglycemia, 2-deoxy-D-glucose (2DG)-induced blockade of glucose utilization, mercaptoacetate (MA)-induced blockade of fatty acid oxidation, 0.9% saline, and 18-h food deprivation were assessed. Unlike unconjugated SAP controls, the DSAP rats did not increase their food intake in response to glucoprivic challenges in either the pelleted food or the intraoral feeding tests. However, the DSAP rats did not differ from SAPs in their ingestive responses to food deprivation and blockade of fatty acid oxidation. The selective impairment of glucoprivic feeding responses indicates that DSAP did not impair the underlying circuitry required for either appetitive or consummatory ingestive responding but eliminated the mechanism for control of this circuitry specifically by glucoprivation. Results suggest that both appetitive and consummatory responses to glucoprivation are controlled and coordinated by multilevel terminations of the same catecholamine neurons.

Prostate cancer invasion and metastasis: insights from mining genomic data.

Prostate cancer (PCa) is the second most commonly diagnosed malignancy in men in the Western world and the second leading cause of cancer-related deaths among men worldwide. Although most cancers have the potential to metastasize under appropriate conditions, PCa favors the skeleton as a primary site of metastasis, suggesting that the bone microenvironment is conducive to its growth. PCa metastasis proceeds through a complex series of molecular events that include angiogenesis at the site of the original tumor, local migration within the primary site, intravasation into the blood stream, survival within the circulation, extravasation of the tumor cells to the target organ and colonization of those cells within the new site. In turn, each one of these steps involves a complicated chain of events that utilize multiple protein-protein interactions, protein signaling cascades and transcriptional changes. Despite the urgent need to improve current biomarkers for diagnosis, prognosis and drug resistance, advances have been slow. Global gene expression methods such as gene microarrays and RNA sequencing enable the study of thousands of genes simultaneously and allow scientists to examine molecular pathways of cancer pathogenesis. In this review, we summarize the current literature that explored high-throughput transcriptome analysis toward the advancement of biomarker discovery for PCa. Novel biomarkers are strongly needed to enable more accurate detection of PCa, improve prediction of tumor aggressiveness and facilitate the discovery of new therapeutic targets for tailored medicine. Promising molecular markers identified from gene expression profiling studies include HPN, CLU1, WT1, WNT5A, AURKA and SPARC.

Titin-based stiffening of muscle fibers in Ehlers-Danlos Syndrome.

OBJECTIVE: tenascin-X (TNX) is an extracellular matrix glycoprotein whose absence leads to Ehlers-Danlos Syndrome (EDS). TNX-deficient EDS patients present with joint hypermobility and muscle weakness attributable to increased compliance of the extracellular matrix. We hypothesized that in response to the increased compliance of the extracellular matrix in TNX-deficient EDS patients, intracellular adaptations take place in the elastic properties of the giant muscle protein titin. METHODS: we performed extensive single muscle fiber mechanical studies to determine active and passive properties in TNX-deficient EDS patients. Gel-electrophoresis, Western blotting, and microarray studies were used to evaluate titin expression and phosphorylation. X-ray diffraction was used to measure myofilament lattice spacing. RESULTS: passive tension of muscle fibers from TNX-deficient EDS patients was markedly increased. Myofilament extraction experiments indicated that the increased passive tension is attributable to changes in the properties of the sarcomeric protein titin. Transcript and protein data indicated no changes in titin isoform expression. Instead, differences in posttranslational modifications within titin's elastic region were found. In patients, active tension was not different at maximal activation level, but at submaximal activation level it was augmented attributable to increased calcium sensitivity. This increased calcium sensitivity might be attributable to stiffer titin molecules. CONCLUSION: in response to the increased compliance of the extracellular matrix in muscle of TNX-deficient EDS patients, a marked intracellular stiffening occurs of the giant protein titin. The stiffening of titin partly compensates for the muscle weakness in these patients by augmenting submaximal active tension generation.

The lipoprivic control of feeding is governed by fat metabolism, not by leptin or adipose depletion.

A lipoprivic control of feeding has been proposed based on the finding that appetite is stimulated by drugs such as beta-mercaptoacetate (MA) that reduce fatty acid oxidation. The adipose-derived hormone, leptin, has effects on feeding and fat oxidation that are opposite those produced by MA. However, effects of this hormone on MA-induced feeding are not known. Here we examined the effects of endogenous leptin levels and of acute central and peripheral leptin administration on MA-induced feeding. We also examined leptin-induced changes in feeding, body weight, and plasma fuels after capsaicin-induced deletion of the lipoprivic control. MA-induced feeding was not altered under any of these conditions, and leptin's effects were not altered by capsaicin. We then examined MA-induced feeding during chronic leptin treatment. Because chronic leptin produces several distinct metabolic states as body adiposity is reduced, we tested MA before, during, and after leptin treatment at times that coincided with these states. MA-induced feeding was unchanged on d 3 of leptin treatment when rats were in a lipolytic state and rapidly metabolizing body fat stores but reduced on d 10 when they were adipose deplete and their level of fat oxidation was reduced. Together results suggest that the lipoprivic control is normally less active in the fat deplete state than during states associated with fat availability. If so, its insensitivity to leptin would enable the lipoprivic control to operate when dietary fat, adiposity, and leptin levels are elevated. The role played by the lipoprivic control under such conditions remains uncertain.

Protein appetite is increased after central leptin-induced fat depletion.

Leptin reduces body fat selectively, sparing body protein. Accordingly, during chronic leptin administration, food intake is suppressed, and body weight is reduced until body fat is depleted. Body weight then stabilizes at this fat-depleted nadir, while food intake returns to normal caloric levels, presumably in defense of energy and nutritional homeostasis. This model of leptin treatment offers the opportunity to examine controls of food intake that are independent of leptin's actions, and provides a window for examining the nature of feeding controls in a "fatless" animal. Here we evaluate macronutrient selection during this fat-depleted phase of leptin treatment. Adult, male Sprague-Dawley rats were maintained on standard pelleted rodent chow and given daily lateral ventricular injections of leptin or vehicle solution until body weight reached the nadir point and food intake returned to normal levels. Injections were then continued for 8 days, during which rats self-selected their daily diet from separate sources of carbohydrate, protein, and fat. Macronutrient choice differed profoundly in leptin and control rats. Leptin rats exhibited a dramatic increase in protein intake, whereas controls exhibited a strong carbohydrate preference. Fat intake did not differ between groups at any time during the 8-day test. Despite these dramatic differences in macronutrient selection, total daily caloric intake did not differ between groups except on day 2. Thus controls of food intake related to ongoing metabolic and nutritional requirements may supersede the negative feedback signals related to body fat stores.