Drastic Ca2+ sensitization of myofilament associated with a small structural change in troponin I in inherited restrictive cardiomyopathy

Six missense mutations in human cardiac troponin I (cTnI) were recently found to cause restrictive cardiomyopathy (RCM). We have bacterially expressed and purified these human cTnI mutants and examined their functional and structural consequences. Inserting the human cTnI into skinned cardiac muscle fibers showed that these mutations had much greater Ca2+-sensitizing effects on force generation than the cTnI mutations in hypertrophic cardiomyopathy (HCM). The mutation K178E in the second actin-tropomyosin (Tm) binding region showed a particularly potent Ca2+-sensitizing effect among the six RCM-causing mutations. Circular dichroism and nuclear magnetic resonance spectroscopy revealed that this mutation does not extensively affect the structure of the whole cTnI molecule, but induces an unexpectedly subtle change in the structure of a region around the mutated residue. The results indicate that the K178E mutation has a localized effect on a structure that is critical to the regulatory function of the second actin-Tm binding region of cTnI. The present study also suggests that both HCM and RCM involving cTnI mutations share a common feature of increased Ca2+ sensitivity of cardiac myofilament, but more severe change in Ca2+ sensitivity is associated with the clinical phenotype of RCM.     

Mac-1(low) early myeloid cells in the bone marrow-derived SP fraction migrate into injured skeletal muscle and participate in muscle regeneration

Recent studies have shown that bone marrow (BM) cells, including the BM side population (BM-SP) cells that enrich hematopoietic stem cells (HSCs), are incorporated into skeletal muscle during regeneration, but it is not clear how and what kinds of BM cells contribute to muscle fiber regeneration. We found that a large number of SP cells migrated from BM to muscles following injury in BM-transplanted mice. These BM-derived SP cells in regenerating muscles expressed different surface markers from those of HSCs and could not reconstitute the mouse blood system. BM-derived SP/Mac-1(low) cells increased in number in regenerating muscles following injury. Importantly, our co-culture studies with activated satellite cells revealed that this fraction carried significant potential for myogenic differentiation. By contrast, mature inflammatory (Mac-1(high)) cells showed negligible myogenic activities. Further, these BM-derived SP/Mac-1(low) cells gave rise to mononucleate myocytes, indicating that their myogenesis was not caused by stochastic fusion with host myogenic cells, although they required cell-to-cell contact with myogenic cells for muscle differentiation. Taken together, our data suggest that neither HSCs nor mature inflammatory cells, but Mac-1(low) early myeloid cells in the BM-derived SP fraction, play an important role in regenerating skeletal muscles.     

Single-nucleotide polymorphism g.1548G > A (E469K) in human ICAM-1 gene affects mRNA splicing pattern and TPA-induced apoptosis

The single-nucleotide polymorphism (SNP) g.1548G > A (E469K) in the human intercellular adhesion molecule-1 (ICAM-1) gene has been suggested to have an association with several types of inflammatory diseases. The polymorphism is located at the three-base position upstream of the splice donor site that produces an alternatively spliced short isoform (ICAM-1-S). To clarify its functional relevance, we studied RNA splicing patterns by comparing cells with different genotype (G/G cells and A/A cells). G/G cells expressed a lower amount of ICAM-1-S mRNA than A/A cells. Since ICAM-1-S has no transmembrane or intracellular domain, ICAM-1 signal transduction and cell-cell contact including Fas-FasL interaction may be influenced. In addition, we studied the effect of this change on FLIP-L mRNA and apoptosis. FLIP-L mRNA tended to decrease, while cell death induced by phorbol 12-myristate 13-acetate was increased. These results suggest that the g.1548 polymorphism modifies inflammatory immune responses by changing cell-cell interaction and then regulating apoptosis.     

The endothelin receptor antagonist ameliorates the hypertensive phenotypes of transgenic hypertensive mice with renin-angiotensin genes and discloses roles of organ specific activation of endothelin system in transgenic mice

Endothelin (ET)-1 and ET-2 are potent vasoconstrictor peptides with mitogenic activity. In this study, we investigated roles of ET system in renin-angiotensin system (RAS)-mediated hypertension, using transgenic hypertensive mice (THM) with over-expression of both human renin and angiotensinogen genes. In the first step, it was revealed that expression of ET system was locally enhanced, i.e. increases in cardiac preproET-1 mRNA and renal preproET-2 mRNA in THM, compared with the control (wild type) mice. In the next step, we studied the chronic effects of an ET antagonist (SB209670) on THM. Blood pressure (BP) in THM was significantly higher than that in the normal mice during the investigation. However, in the later phase of the study, from 12 to 20 weeks of treatment, THM receiving SB 209670 showed significantly lower BP than that in THM receiving saline. SB 209670 treatment for 20 weeks significantly attenuated phenotypes of cardiac hypertrophy, vascular wall thickening and hypertensive nephropathy observed in THM, suggesting that the ETA/B receptor antagonist is also effective even in the extraordinarily activated RAS condition. These findings suggest that organ specifically activated ET system in THM develops the phenotypes, hypertension, cardiac hypertrophy, and hypertensive nephropathy.     

Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a β-carotene 3-hydroxylase/4-ketolase

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a β-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the β-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this β-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from β-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of β-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both β-ionone ends.     

EPA effect on NOS gene expression and on NO level in endothelin-1-induced hypertrophied cardiomyocytes

Cardiomyocytes release (or metabolize) several diffusible agents (e.g., nitric oxide [NO], endothelin-1 [ET-1], and angiotensin II) that exert direct effects on myocyte function under various pathologic conditions. Although cardiac hypertrophy is a compensatory mechanism in response to different cardiovascular diseases, there can be a pathologic transition in which the myocardium becomes dysfunctional. Recently, NO has been found to be an important regulator of cardiac remodeling. Specifically, NO has been recognized as a potent antihypertrophic and proapoptotic mediator in cultured cardiomyocytes. We demonstrated that ET-1-induced hypertrophic remodeling in neonatal cardiomyocytes was arrested by pretreatment with eicosapentaenoic acid (EPA), a major component of fish oil. In some recent studies, EPA has demonstrated cardioprotective effects by modulating NO. This study investigated the changes in NO synthase (NOS) in ET-1-induced hypertrophied cardiomyocytes and in total levels of nitrates and nitrites. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured in D-MEM/Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control group, ET-1 (0.1 nM) group, and ET-1 pretreated with EPA (10 microM) group. NOS gene expression was evaluated 24 hrs after treatment using real-time polymerase chain reaction. Endothelial NOS (eNOS) mRNA expression was decreased in the ET-1 group compared with controls and was unchanged by pretreatment with EPA. mRNA expression of inducible NOS (iNOS) was significantly increased in ET-1-treated cardiomyocytes and was suppressed by EPA pretreatment. Neuronal NOS gene expression and total NO level did not exhibit a statistically significant change in any of the groups. There may be some interaction between ET-1, eNOS, and iNOS in ET-1-induced and EPA-regressed hypertrophied cardiomyocytes that suppress iNOS expression without modulating total NO level or eNOS gene expression.     

Alterations of gene expressions of preproET-1 and ET receptors in brains of endotoxemic Sprague-Dawley rats

During severe sepsis, several immunological defense mechanisms initiate a cascade of inflammatory events leading to multiorgan failure, including septic encephalopathy and ultimately death. Endothelin-1 (ET-1) has recently been investigated in different cerebral pathologies. Some reports suggest the involvement of ET-1 in sepsis. However, no study to date has reported the alterations in expression of the genes encoding preproET-1 and ET receptors in the frontal cortex of the septic brain. Male Sprague-Dawley (SD) rats 8 weeks of age were administered either saline or 15 mg/kg lipopolysaccharide (LPS) at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. The rats were sacrificed with ether, and the brain tissues were harvested. Systolic and diastolic blood pressure decreased 1 hr after LPS administration and then gradually returned to normal, without any change in the heart rate. We confirmed the induction of endotoxemia in the brains of SD rats by measuring the expression of nitric oxide synthase (NOS) mRNA induced in the cerebrum. The expression of inducible NOS (iNOS) mRNA in the brains of SD rat after LPS administration was 30-fold higher than that in the brains of control rats. mRNA expression of preproET-1 in the frontal cortex of SD rats after LPS administration was 2-fold higher than that in control rats. A time-dependent increase in the expression of the gene encoding the ET(A) receptor (vasoconstrictive property) after LPS administration was observed in SD rat brain, whereas expression of the gene encoding the ET(B) receptor (vasodilatatory property) showed an initial upregulation and then gradually decreased as sepsis progressed. In conclusion, we report for the first time that expressions of the genes encoding ET-1 and ET receptors are altered in the endotoxemic brain and that these alterations are time-dependent in SD rats. The alterations in the ET system in brain tissue observed in the present study may contribute to the understanding of the pathophysiological changes in the endotoxemic brain.     

Changes in important apoptosis-related molecules in the endothelin-1-induced hypertrophied cardiomyocytes: effect of the pretreatment with eicosapentaenoic Acid

Human heart failure is preceded by a process called cardiac remodeling, in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Endothelin (ET)-1, a potent vasoconstrictor peptide, is synthesized and secreted by cardiomyocytes and induces hypertrophy of cardiomyocytes. The cardiovascular benefit of fish oil containing eicosapentaenoic acid (EPA) in humans and experimental animals was reported. Recently, we found that ET-1-induced cardiomyocytic remodeling could be prevented by pretreatment with EPA. The aim of the present study is to investigate whether there would be any alteration in the expression of important apoptosis-related molecules in ET-1-administered hypertrophied cardiomyocytes. We also sought to determine, if there are alterations in apoptotic molecules, what type of role for EPA would then exist. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control, the ET-1 (0.1 nM)-treated group, and the ET-1 group pretreated with EPA (10 microM). Twenty-four hours after the treatment, the gene expressions of three important molecules related to apoptosis (caspase-3, Bax, and Bcl-2) in three experimental groups were evaluated by real-time polymerase chain reaction. The present study could not demonstrate any significant or representative alteration in any of the above three apoptosis-related important markers in either ET-1-induced hypertrophied cardiomyocytes with or without EPA pretreatment. The present study would at least be able to exclude the involvement of some representative molecules related to apoptosis in ET-1-induced hypertrophied cardiomyocytes. In addition, the present study demonstrates that the antihypertrophic effect of EPA to ET-1-administered cardiomyocytes appears not to modulate the apoptosis signaling cascade.     

Oxidation of Arg-410 promotes the elimination of human serum albumin

The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs. The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice after labeling with 111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase in carbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in alpha-helical content as the result of oxidation was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in the pharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A and R410A mutants were affected (extent of effect: R410A > K199A > H146A). The liver clearance of these proteins is closely correlated to hydrophobicity (r = 0.929, P < 0.01) and the net charge of the proteins (r=0.930, P < 0.01). The rate of elimination of HSA is closely related to the hydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.