[Abstract unavailable

Abstract-The importance of proteasomes in governing the intracellular protein degradation process has been increasingly recognized. Recent investigations indicate that proteasome complexes may exist in a species-and cell-type-specific fashion. To date, despite evidence linking impaired protein degradation to cardiac disease phenotypes, virtually nothing is known regarding the molecular composition, function, or regulation of cardiac proteasomes. We have taken a functional proteomic approach to characterize 26S proteasomes in the murine heart. Multidimensional chromatography was used to obtain highly purified and functionally viable cardiac 20S and 19S proteasome complexes, which were subjected to electrophoresis and tandem mass spectrometry analyses. Our data revealed complex molecular organization of cardiac 26S proteasomes, some of which are similar to what were reported in yeast, whereas others exhibit contrasting features that have not been previously identified in other species or cell types. At least 36 distinct subunits (17 of 20S and 19 of 19S) are coexpressed and assembled as 26S proteasomes in this vital cardiac organelle, whereas the expression of PA200 and 11S subunits were detected with limited participation in the 26S complexes. The 19S subunits included a new alternatively spliced isoform of Rpn10 (Rpn10b) along with its primary isoform (Rpn10a). Immunoblotting and immunocytochemistry verified the expression of key ␣ and ␤ subunits in cardiomyocytes. The expression of 14 constitutive ␣ and ␤ subunits in parallel with their three inducible subunits (␤1i, ␤2i, and ␤5i) in the normal heart was not expected; these findings represent a distinct level of structural complexity of cardiac proteasomes, significantly different from that of yeast and human erythrocytes. Furthermore, liquid chromatography/tandem mass spectroscopy characterized 3 distinct types of post-translational modifications including (1) N-terminal acetylation of 19S subunits (Rpn1, Rpn5, Rpn6, Rpt3, and Rpt6) and 20S subunits (␣2, ␣5, ␣7, ␤3, and ␤4); (2) N-terminal myristoylation of a 19S subunit (Rpt2); and (3) phosphorylation of 20S subunits (eg, ␣7)). Taken together, this report presents the first comprehensive characterization of cardiac 26S proteasomes, providing critical structural and proteomic information fundamental to our future understanding of this essential protein degradation system in the normal and diseased myocardium. T he proteasome is a key proteolytic enzymatic system governing the degradation of majority intracellular proteins. 1 Recent studies implicate proteasomes in cardiac diseases. [2][3][4][5][6][7][8][9][10][11] Proteasome inhibitors are widely used in cancer therapy, however, their impact on cardiac function remains unclear; investigations in the heart have reported conflicting results. 2-4,12 A major contributing factor to these controversies is the lack of information pertaining to the structural organization and protein composition of cardiac proteasomes, therefore prohibiting the identification of mol...

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca 2؉ binding site, had dramatically altered Ca 2؉ binding properties. The K Ca value for E22K was decreased by ϳ17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca 2؉ . Interestingly, Ca 2؉binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the ␣-helical content of phosphorylated R58Q greatly increased with Ca 2؉ binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K Ca than wild-type light chain, whereas phosphorylation of this mutant increased the Ca 2؉ affinity 6-fold. Whereas phosphorylation of wildtype light chain decreased its Ca 2؉ affinity, the opposite was true for A13T. The ␣-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca 2؉ binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.There is substantial evidence that myosin regulatory light chains (RLC) 1 play a primary regulatory role in scallop and smooth muscle contraction, but their functional role in mammalian striated (skeletal and cardiac) muscle contraction is unclear. RLC, together with the essential light chain, stabilizes the 8.5-nm-long ␣-helical neck of the myosin head, with the N terminus of RLC wrapped around the heavy chain (1). Smooth muscle contraction is initiated by RLC phosphorylation with a Ca 2ϩ -calmodulin-activated myosin light chain kinase (MLCK) (2, 3). However, in skeletal and cardiac muscle, RLC phosphorylation does not activate contraction but appears to play a modulatory role (4). It was shown that RLC phosphorylation increased the Ca 2ϩ sensitivity of force in skinned skeletal (5-7) and cardiac (8) muscle fibers. In the human heart, several RLC isoforms are expressed (9, 10) preferentially in the atrium and in the ventricle. Recent studies have revealed that the ventricular RLC is one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (11,12). FHC is an autosomal dominant disease, characterized by left ventricular hypertrophy, myofibrillar disarray, and sudden death. It is caused by missense mutations in various genes that encode for ␤-myosin heavy chain (13), myosin-binding protein C (14), ventricular RLC and essential light chain (11,12,15), troponin T (16), troponin I (17), ␣-tropomyosin (18), actin (19), and titin (20). Depending on the affected gene, and the site of the mutation, FHC has variable presentation with regard to its degree and severity and t...

Key pointsr Ribosome biogenesis is the primary determinant of translational capacity, but its regulation in skeletal muscle following acute resistance exercise is poorly understood.r Resistance exercise increases muscle protein synthesis acutely, and muscle mass with training, but the role of translational capacity in these processes is unclear.r Here, we show that acute resistance exercise activated pathways controlling translational activity and capacity through both rapamycin-sensitive and -insensitive mechanisms.r Transcription factor c-Myc and its downstream targets, which are known to regulate ribosome biogenesis in other cell types, were upregulated after resistance exercise in a rapamycin-independent manner and may play a role in determining translational capacity in skeletal muscle.r Local inhibition of myostatin was also not affected by rapamycin and may contribute to the rapamycin-independent effects of resistance exercise.Abstract This study aimed to determine (1) the effect of acute resistance exercise on mechanisms of ribosome biogenesis, and (2) the impact of mammalian target of rapamycin on ribosome biogenesis, and muscle protein synthesis (MPS) and degradation. Female F344BN rats underwent unilateral electrical stimulation of the sciatic nerve to mimic resistance exercise in the tibialis anterior (TA) muscle. TA muscles were collected at intervals over the 36 h of exercise recovery (REx); separate groups of animals were administered rapamycin pre-exercise (REx+Rapamycin). Resistance exercise led to a prolonged (6-36 h) elevation (30-50%) of MPS that was fully blocked by rapamycin at 6 h but only partially at 18 h. REx also altered pathways that regulate protein homeostasis and mRNA translation in a manner that was both rapamycin-sensitive (proteasome activity; phosphorylation of S6K1 and rpS6) and rapamycin-insensitive (phosphorylation of eEF2, ERK1/2 and UBF; gene expression of the myostatin target Mighty as well as c-Myc and its targets involved in ribosome biogenesis). The role of c-Myc was tested in vitro using the inhibitor 10058-F4, which, over time, decreased basal RNA and MPS in a dose-dependent manner (correlation of RNA and MPS, r 2 = 0.98), even though it had no effect on the acute stimulation of protein synthesis. In conclusion, acute resistance exercise stimulated rapamycin-sensitive and -insensitive mechanisms that regulate translation activity and capacity.

Abstract-Our recent studies have provided a proteomic blueprint of the 26S proteasome complexes in the heart, among which 20S proteasomes were found to contain cylinder-shaped structures consisting of both ␣ and ␤ subunits. These proteasomes exhibit a number of features unique to the myocardium, including striking differences in post-translational modifications (PTMs) of individual subunits and novel PTMs that have not been previously reported. To date, mechanisms contributing to the regulation of this myocardial proteolytic core system remain largely undefined; in particular, little is known regarding PTM-dependent regulation of cardiac proteasomes. In this investigation, we seek to elucidate the function and regulation of 20S proteasome complexes in the heart. Functionally viable murine cardiac 20S proteasomes were purified. Tandem mass spectrometry analyses, combined with native gel electrophoresis, immunoprecipitation, and immunoblotting, revealed the identification of 2 previously unrecognized functional partners in the endogenous intact cardiac 20S complexes: protein phosphatase 2A (PP2A), and protein kinase A (PKA). Furthermore, our results demonstrated that PP2A and PKA profoundly impact the proteolytic function of 20S proteasomes: phosphorylation of 20S complexes enhances the peptidase activity of individual subunits in a substrate-specific fashion. Moreover, inhibition of PP2A or the addition of PKA significantly modified both the serine-and threonine-phosphorylation profile of proteasomes; multiple individual subunits of 20S (eg, ␣1 and ␤2) were targets of PP2A and PKA. Taken together, these studies provide the first demonstration that the function of cardiac 20S proteasomes is modulated by associating partners and that phosphorylation may serve as a key mechanism for regulation.

At least four isoforms of troponin T (TnT) exist in the human heart, and they are expressed in a developmentally regulated manner. To determine whether the different N-terminal isoforms are functionally distinct with respect to structure, Ca 2؉ sensitivity, and inhibition of force development, the four known human cardiac troponin T isoforms, TnT1 (all exons present), TnT2 (missing exon 4), TnT3 (missing exon 5), and TnT4 (missing exons 4 and 5), were expressed, purified, and utilized in skinned fiber studies and in reconstituted actomyosin ATPase assays. TnT3, the adult isoform, had a slightly higher ␣-helical content than the other three isoforms. The variable region in the N terminus of cardiac TnT was found to contribute to the determination of the Ca 2؉ sensitivity of force development in a charge-dependent manner; the greater the charge the higher the Ca 2؉ sensitivity, and this was primarily because of exon 5. These studies also demonstrated that removal of either exon 4 or exon 5 from TnT increased the cooperativity of the pCa force relationship. Troponin complexes reconstituted with the four TnT isoforms all yielded the same maximal actin-tropomyosin-activated myosin ATPase activity. However, troponin complexes containing either TnT1 or TnT2 (both containing exon 5) had a reduced ability to inhibit this ATPase activity when compared with wild type troponin (which contains TnT3). Interestingly, fibers containing these isoforms also showed less relaxation suggesting that exon 5 of cardiac TnT affects the ability of Tn to inhibit force development and ATPase activity. These results suggest that the different N-terminal TnT isoforms would produce different functional properties in the heart that would directly affect myocardial contraction.

This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine ␣-myosin heavy chain promoter. Extensive characterization of the transgenic I79N lines compared with wild type and/or nontransgenic mice demonstrated: 1) normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca 2؉ sensitivity of ATPase activity and force in skinned fibers; 3) a substantial increase in the rate of force activation and an increase in the rate of force relaxation; 4) lower maximal force/cross-sectional area and ATPase activity; 5) loss of sensitivity to pHinduced shifts in the Ca 2؉ dependence of force; and 6) computer simulations that reproduced experimental observations and suggested that the I79N mutation decreases the apparent off rate of Ca 2؉ from troponin C and increases cross-bridge detachment rate g. Simulations for intact living fibers predict a higher basal contractility, a faster rate of force development, slower relaxation, and increased resting tension in transgenic I79N myocardium compared with transgenic wild type. These mechanisms may contribute to mortality in humans, especially in stimulated contractile states.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs worldwide. NSAIDs are used for a variety of conditions including pain, rheumatoid arthritis, and musculoskeletal disorders. The beneficial effects of NSAIDs in reducing or relieving pain are well established, and other benefits such as reducing inflammation and anticancer effects are also documented. The undesirable side effects of NSAIDs include ulcers, internal bleeding, kidney failure, and increased risk of heart attack and stroke. Some of these side effects may be due to the oxidative stress induced by NSAIDs in different tissues. NSAIDs have been shown to induce reactive oxygen species (ROS) in different cell types including cardiac and cardiovascular related cells. Increases in ROS result in increased levels of oxidized proteins which alters key intracellular signaling pathways. One of these key pathways is apoptosis which causes cell death when significantly activated. This review discusses the relationship between NSAIDs and cardiovascular diseases (CVD) and the role of NSAID-induced ROS in CVD.