Isoform In Heart Research Articles

Molecular and Functional Characterization of a Novel Cardiac-Specific Human Tropomyosin Isoform

Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1alpha (also called alpha-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1kappa compared with TPM1alpha. This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

Abstract 5787: Discovery and Characterisation of Linear and Cyclic Peptide Agonists of Apelins

The recently discovered apelin family of peptides mediate their actions by a single G-protein coupled receptor, APJ. We have previously shown apelins have three major actions in the human cardiovascular system: endothelium-dependent vasodilatation; direct vasoconstriction by interacting with smooth muscle APJ receptor and increased cardiac contractility by action on cardiac myocytes. [Pyr 1 ]apelin-13 was identified as the predominant isoform in human heart. Our aim was to discover shorter sequences of [Pyr 1 ]apelin-13 retaining agonist activity and use a computational ligand based strategy to design cyclic peptides. Over 30 compounds were synthesised and tested in a competition binding assay against [Glp65,Nle75,Tyr77] [ 125 I]Apelin-13. Functional assays measured the vaso-constrictor action in endothelium denuded human saphenous veins in oxygenated Krebs’ solution (37°C) in organ baths for isometric tension recordings. Cumulative concentration response curves were constructed to the endogenous and synthetic analogues. Responses were expressed as percentage of the terminal KCl (0.1M) constriction. Data are expressed as mean ± S.E. Mean and n-values are the number of patients from whom tissues were obtained. One micromolar concentrations of the predicted cleavage product of the angiotensin converting enzyme 2 (ACE-2), apelin-13(1–12), inhibiting radiolabelled apelin binding by 60.0±7.0% (n=3). The most potent cyclic analogue identified, MM07 inhibited binding by 42.2±4.8% (n=3) compared with 76.0±4.5% inhibition by (Pyr 1 )apelin-13. Both compounds potently constricted endothelium denuded saphenous vein (Apelin-13(1–12): pD 2 9.07±0.40, E max 29.30±9.43% KCl, n=5; MM07: pD 2 10.53±0.24, E max 21.80±5.72% KCl, n=3) with a comparable potency and efficacy to (Pyr 1 )apelin-13 (pD2 (pD 2 = 8.8±0.3, E MAX = 26±4%, n=15). These results show unexpectedly that the cleavage product of ACE-2 retains full agonist activity. The novel cyclic peptide retains potency and can be used as a pharmacological tool to characterise the apelin system and in the design of selective antagonists.

Triadin, not essential, but useful

Triadin, not essential, but useful

Discovery of lipoprotein lipase pI isoforms and contributions to their characterization

Lipoprotein lipase (LPL) plays a pivotal role in lipid metabolism and is implicated in several pathophysiological conditions. A large number of LPL studies have been performed in rat, although the amount of information derived from direct study of the protein in this species is limited. Here we attempted to examine possible modifications of LPL using proteomic tools. By combining high-resolution two-dimensional gel electrophoresis and Western blot with biological mass spectrometry we demonstrate the coexistence of multiple LPL pI isoforms in rat heart. We studied the origin of this pI heterogeneity by: (1) comparison with the 2D pattern of LPL from post-heparin rat plasma (as a source of mature LPL); (2) protein dephosphorylation; (3) protein deglycosylation; and (4) partial sequencing of LPL isoforms. The results reveal that LPL pI heterogeneity does not correspond to different stages of intracellular maturation or protein phosphorylation. It can be partially explained by glycosylation, although other post-translational modifications must also be involved. We also report the first partial sequence to be obtained from direct study of rat LPL protein. These findings should be the basis for further research aimed at identifying the molecular differences between LPL isoforms and exploring their potential functional implications.

Characterization of Mouse Heart Adenylyl Cyclase

Chronic heart failure is one of the most frequent causes of death in humans. Knockout of type 5 adenylyl cyclase (AC) in mice causes longevity and protection from cardiomyopathy, and an AC5 inhibitor reduces beta-adrenoceptor-stimulated Ca(2+) inward currents in isolated mouse cardiomyocytes. These data indicate that selective AC5 inhibitors may be beneficial in chronic heart failure. Therefore, we characterized AC in mouse heart membranes. Real-time polymerase chain reaction and immunoblot analysis suggested that AC5 is an important heart AC isoform. Enzyme kinetics of heart AC and recombinant AC5 in the presence of Mg(2+) were similar. Moreover, the inhibitory profile of eight 2'(3')-O-(N-methylanthraniloyl) (MANT)-nucleoside 5'-([gamma-thio])triphosphates on mouse heart in the presence of Mg(2+) was almost identical to that of AC5. MANT-ITP was the most potent inhibitor of heart AC and recombinant AC5, with K(i) values in the 15 to 25 nM range in the presence of Mg(2+) and in the 1 to 5 nM range in the presence of Mn(2+). However, in the presence of Mn(2+), we also noted differences between mouse heart AC and AC5 with respect to enzyme kinetics and forskolin analog effects. In conclusion, with regard to expression and kinetics and inhibition by MANT-nucleotides in the presence of Mg(2+), AC5 is an important AC isoform in heart, with MANT-ITP being an excellent starting point for the design of AC5-selective inhibitors. Unfortunately, a limitation of our study is the fact that immunologically and biochemically, AC5 and AC6 are quite similar, although they have different roles in heart. Moreover, lack of antibody specificity and Mn(2+) masking AC5 effects were problems.

Role of L1462 in Nav1.5 Channel DIII-S6 in Voltage-dependent Gating and Antiarrhythmic Block

Mutations of domain III S6 (DIII-S6) Leu-1465 of rat brain IIA Na+ channel (Nav1.2) or the equivalent (L1280) in the adult rat skeletal muscle isoform (Nav1.4) affect local anesthetic and anticonvulsant block (Yarov-Yarovoy et al., 2001; Nau et al., 2003). We examined the role of the equivalent position (L1462) of the human heart isoform (Nav1.5) in voltage-dependent gating and antiarrhythmic block. Whole-cell Na+ currents were measured in HEK293 cells transiently expressing the recombinant wild type (WT) or mutant channels and the beta-1 subunit. Compared to WT, all the mutants of L1462 (L1462A, L1462C, L1462F) accelerated the current decay and shifted the voltage dependence of activation towards more positive direction. L1462 mutants had no effects on the voltage dependence of fast inactivation and the recovery from fast inactivation, but increased the fraction of intermediate inactivation component. Internal charged methanethiosulfonate, MTSES blocked L1462C/C373F with a high frequency of stimulation (20 Hz), where C373 is known to face the outer pore and be the critical residue for isoform differences in tetrodotoxin block. L1462F had no obvious effects on tonic block, but increased use-dependent block by the class Ic antiarrhythmic drug, flecainide, which binds preferentially to activated channels. L1462F increased affinity of activated channels for flecainide by 4-fold, while L1462F had little effects on affinity of inactivated channels. For internal membrane-impermeant QX314, L1462F increased use-dependent block and affinity for QX314 binding to activated channels by 5-fold. These results suggest that L1462 faces the pore in the open state and is involved in activated channel block by antiarrhythmic drugs as well as inactivated channel block by local anesthetics.

β3-Adrenergic Receptor Subtype Signaling In Senescent Heart

β3-Adrenergic Receptor Subtype Signaling In Senescent Heart

Voltage-gated Na+ channel transcript patterns in the mammalian heart are species-dependent

In the present study we investigated whether transcription, alternative splicing and developmental regulation of voltage-gated Na+ channels occur in a species-dependent manner in the mammalian heart. The composition of the Na+ channel transcript pool including Nav1.1–Nav1.5 was analysed by RT-PCR in mouse, rat, pig, and human hearts. We found that relative transcript levels of tetrodotoxin (TTX) sensitive channels (Nav1.1–Nav1.4) decreased with increasing heart size (30% for mouse, 8% for rat, and 4% for pig/human). Considering transcript levels of individual isoforms, human and pig hearts were nearly indistinguishable whereas large differences existed between human and mouse. We also noticed that alternative splicing and age-dependent Na+ channel expression occurred in a species-dependent manner. Unexpectedly, we even observed gender differences in the cardiac levels of TTX sensitive Na+ channels in humans. Our data suggest that species differences could underlie published discrepancies on the role of TTX sensitive Na+ channels in the heart and that the functions of those minor cardiac isoforms in normal and diseased hearts are best studied in larger mammalian animals.

What Are the Consequences of Phosphorylation and Hyperphosphorylation of Ryanodine Receptors in Normal and Failing Heart?

See related article, pages e65–e72 Marks and colleagues published some influential results in 2000, suggesting that protein kinase A phosphorylation regulates ryanodine receptor (RyR2) open probability ( P oRyR). The authors attributed this to the dissociation of the RyR-associated regulatory protein FKBP12.6.1 They went on to propose that, in failing hearts, RyR2 is hyperphosphorylated, which leads to defective channel function by increasing sensitivity to Ca-induced activation of the molecule. Moreover, these effects were attributable to phosphorylation of a single amino acid, serine 2809 (S2809). There are 4 of these serine molecules at location 2808 in the human RyR2 molecule, 1 on each subunit of the tetramer. Much subsequent work has referred to the 2809 residue, which is found in rabbits. Marks and colleagues further suggested that phosphorylation of S2809 was also responsible for the normal effects of adrenergic stimulation on excitation–contraction coupling (ECC) and that hyperphosphorylation was responsible for abnormal ECC during failure. The RyR is a tetramer that forms the largest membrane ion channel. In muscle, it is found in the sarcoplasmic reticulum (SR), where it functions as the Ca release channel. The heart isoform is designated RyR2. The pore sequences are largely found in membrane spanning domains.2 There is a large cytosolic domain. Although hyperphosphorylation refers, by definition, to the occupation of all the putative phosphorylation sites in a molecule in the context of the work by Marks and colleagues,1 hyperphosphorylation refers to 3 or perhaps 4 of the serine residues. In RyRs, there are a large number of consensus phosphorylation sites.3 RyRs are associated with a number of proteins, and these include anchoring protein phosphatases and kinases, as well as the regulatory protein FKBP12.6. From the standpoint of this editorial, this last protein is perhaps the most interesting, and it is considered …

Enzymatic properties of the lactate dehydrogenase enzyme from Plasmodium falciparum

The lactate dehydrogenase enzyme from Plasmodium falciparum (PfLDH) is a target for antimalarial compounds owing to structural and functional differences from the human isozymes. The plasmodial enzyme possesses a five-residue insertion in the substrate-specificity loop and exhibits less marked substrate inhibition than its mammalian counterparts. Here we provide a comprehensive kinetic analysis of the enzyme by steady-state and transient kinetic methods. The mechanism deduced by product inhibition studies proves that PfLDH shares a common mechanism with the human LDHs, that of an ordered sequential bireactant system with coenzyme binding first. Transient kinetic analysis reveals that the major rate-limiting step is the closure of the substrate-specificity loop prior to hydride transfer, in line with other LDHs. The five-residue insertion in this loop markedly increases substrate specificity compared with the human muscle and heart isoforms.

Expression and transcriptional activity of alternative splice variants of Mitf exon 6

Microphthalmia-associated transcription factor (Mitf) is a tissue-specific transcription factor. At least nine distinct mouse isoform mRNAs are encoded by alternative splicing of the first exon of Mitf (Mitf-A, -B, -C, -D, -E, -H, -J, -M, and -mc), while exons 2-9 of all Mitf isoforms examined to date are identical. In addition, alternative splice variants of exon 6a encoding 6 amino acid proximal to the basic region of the protein are known in Mitf-A, -H, and -M. In this study, we identified alternative splice variants of exon 6a in other Mitf isoforms (Mitf-E, -J, and -mc) in melanocytes, mast cells, macrophages, and heart. We also compared the transcriptional activity of Mitf variants containing exon 6a to that of Mitf variants that did not contain exon 6a. PCR-RFLP analysis revealed that expression of Mitf with exon 6a was comparable with that of Mitf without exon 6a, irrespective of the specificity of the first exon, or cell type, although Mitf isoforms with different first exons were expressed in a cell type-dependent manner. Luciferase-based reporter assays revealed that transcription of Tyrosinase, which is known Mitf-regulated gene, was elicited more efficiently by expression of Mitf isoforms containing exon 6a, compared to isoforms that did not contain exon 6a. However, when transcription of Tyrp-1, Mmcp-6, and PAI-1 was examined, no significant differences were detected between Mitf isoforms with exon 6a and those without exon 6a, except for Tyrp-1 transcription by Mitf-D/E isoform. These results reveal a diverse pattern of gene expression and different transcriptional activities of Mitf isoforms, suggesting discrete regulation of gene transcription in specific tissues by Mitf.

Role of myofibril-inducing RNA in cardiac TnT expression in developing Mexican axolotl

The Mexican axolotl, Ambystoma mexicanum, has been a useful animal model to study heart development and cardiac myofibrillogenesis. A naturally-occurring recessive mutant, gene ``c”, for cardiac non-function in the Mexican axolotl causes a failure of myofibrillogenesis due to a lack of tropomyosin expression in homozygous mutant (c/c) embryonic hearts. Myofibril-inducing RNA (MIR) rescues mutant hearts in vitro by promoting tropomyosin expression and myofibril formation thereafter. We have studied the effect of MIR on the expression of various isoforms of cardiac troponin T (cTnT), a component of the thin filament that binds with tropomyosin. Four alternatively spliced cTnT isoforms have been characterized from developing axolotl heart. The expression of various cTnT isoforms in normal, mutant, and mutant hearts corrected with MIR, is evaluated by real-time RT-PCR using isoform specific primer pairs; MIR affects the total transcription as well as the splicing of the cTnT in axolotl heart.

A novel signaling pathway for β-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes

Stimulation of cardiac beta-adrenergic receptors (beta-AR) activates both the G(s)- and G(i)-coupled signaling cascades, including the phosphoinositide 3 kinase (PI3K) pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking beta-AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute beta-AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Cotreatment with ICI-118,551, but not CGP-20712, abolished the increase in PI3K activity, suggesting a beta(2)-AR-mediated event. PI3K activation was also abrogated by cotreatment with pertussis toxin, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2, a selective Src-family tyrosine kinases inhibitor), or AG-1296 [selective platelet-derived growth factor receptor (PDGFR) inhibitor] but not with an inhibitor for protein kinase A, protein kinase C, Ras, adenylyl cyclase, epidermal growth factor receptor, or insulin-like growth factor-1 receptor. beta-AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or small interfering RNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gbetagamma sequestrant peptide derived from the COOH-terminus of beta-AR kinase-1 failed to activate PI3K after beta-AR stimulation, suggesting Gbetagamma is required for the transactivation. Furthermore, acute beta-AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3Kalpha isoform activities but not the activities of other isoforms (PI3Kbeta, -delta, -gamma) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of beta-AR-mediated transactivation of cardiac PI3Kalpha via sequential involvement of Galpha(i)/Gbetagamma, Src, and PDGFR.

Ca2+ Activation Kinetics of the Two Aspartate-Glutamate Mitochondrial Carriers, Aralar and Citrin: ROLE IN THE HEART MALATE-ASPARTATE NADH SHUTTLE

Ca(2+) regulation of the Ca(2+) binding mitochondrial carriers for aspartate/glutamate (AGCs) is provided by their N-terminal extensions, which face the intermembrane space. The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle. We report that their N-terminal extensions contain up to four pairs of EF-hand motifs plus a single vestigial EF-hand, and have no known homolog. Aralar and citrin contain one fully canonical EF-hand pair and aralar two additional half-pairs, in which a single EF-hand is predicted to bind Ca(2+). Shuttle activity in brain or skeletal muscle mitochondria, which contain aralar as the major AGC, is activated by Ca(2+) with S(0.5) values of 280-350 nm; higher than those obtained in liver mitochondria (100-150 nm) that contain citrin as the major AGC. We have used aralar- and citrin-deficient mice to study the role of the two isoforms in heart, which expresses both AGCs. The S(0.5) for Ca(2+) activation of the shuttle in heart mitochondria is about 300 nm, and it remains essentially unchanged in citrin-deficient mice, although it undergoes a drastic reduction to about 100 nm in aralar-deficient mice. Therefore, aralar and citrin, when expressed as single isoforms in heart, confer differences in Ca(2+) activation of shuttle activity, probably associated with their structural differences. In addition, the results reveal that the two AGCs fully account for shuttle activity in mouse heart mitochondria and that no other glutamate transporter can replace the AGCs in this pathway.

The complete nuclear estrogen receptor family in the rainbow trout: Discovery of the novel ERα2 and both ERβ isoforms

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERα (NR3A1) and ERβ (NR3A2). The ERβ subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERα have not been demonstrated in any fish species. In the rainbow trout ( Oncorhynchus mykiss), the only ER described is an isoform of the ERα subtype (i.e. ERα1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERα isoform, ERα2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERα1. Both the rainbow trout ERβ subtypes, ERβ1 [NR3A2a] and ERβ2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERαs (∼ 40% similarity). ERβ1 and ERβ2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERα subtype clade, with the closest relative to rainbow trout ERα2 being rainbow trout ERα1, suggesting a recent, unique duplication event to create these two isoforms. For the ERβ subtype there are two distinct subclades, one represented by the ERβ1 isoform and the other by the ERβ2 isoform. The rainbow trout ERβ1 and ERβ2 are not closely associated with each other, but instead fall into their respective ERβ subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms ( ERα1, ERα2, ERβ1, and ERβ2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.

A novel signaling pathway for β‐adrenergic receptor‐mediated activation of PI3K in H9c2 cardiomyocytes

Stimulation of cardiac β-adrenergic receptors (βAR) activates both Gs-and Gi-coupled signaling cascades, including the PI3K pathway, that have important physiological implications. Multiple isoforms of PI3K are found in the heart. The goals of the current study were to examine the intracellular signaling pathways linking βAR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute βAR stimulation with isoproterenol (ISO) resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Co-treatment with ICI 118,551, but not CGP 20712, abolished ISO-induced increase in PI3K activity, suggesting a β2AR-mediated event. The increase in PI3K activity was also abrogated by co-treatment with PP2, a selective inhibitor of the Src-family tyrosine kinases, and by AG 1296, a selective inhibitor of PDGFR, but not by inhibitors of Gαi, PKA, PKC, Ras, adenylyl cyclase, EGFR or IGF-1R. Consistent with these results, βAR-induced increases in tyrosine phosphorylation of PDGFR and Src, which can be abolished by siRNA specific for Src. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gβγ sequestrant peptide derived from the C-terminus of βAR kinase-1 failed to activate PI3K after βAR stimulation, suggesting Gβγ is required for the transactivation. More important, acute βAR stimulation in vivo resulted in increases in tyrosine kinase-sensitive PI3K and PI3Kα activities but not the activities of other isoforms (PI3Kβ, -δ, -γ) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of βAR-mediated transactivation of cardiac PI3Kα via sequential involvement of Gβγ, Src and PDGFR. (Supported by NIH 1 P20 RR018728)

Recombinase‐mediated cassette exchange to rapidly and efficiently generate mice with human cardiac sodium channels

SCN5A encodes the predominant voltage-gated sodium channel isoform in human heart and nearly 100 variants have now been described and studied in vitro. However, development of animal models to analyze function of such large numbers of human gene variants represents a continuing challenge in translational medicine. Here, we describe the implementation of a two stage procedure, recombinase-mediated cassette exchange (RMCE), to efficiently and rapidly generate mice in which a full-length human cDNA replaces expression of the murine ortholog. In the first step of RMCE, conventional homologous recombination in mouse ES cells was used to replace scn5a exon 2 (that contains the translation start site) with a cassette acceptor that includes the thymidine kinase gene, flanked by loxP/inverted loxP sites. In the second step, the cassette acceptor site was replaced by the full-length wild-type human SCN5A cDNA by Cre/loxP-mediated recombination. The exchange event occurred in 7/29 (24%) colonies, and the time from electroporation to first homozygotes was only 8 months. PCR-restriction fragment length polymorphism (RFLP) showed that the murine isoform was replaced by the human one, and functional studies indicated that mice with human cardiac sodium channels have wild-type sodium current density, action potential durations, heart rates, and QRS durations. These data demonstrate that RMCE can be used to generate mice in which a targeted allele can be rapidly and efficiently replaced by variants of choice, and thereby can serve as an enabling approach for the functional characterization of ion channel and other DNA variants.

Transcription factor MITF regulates cardiac growth and hypertrophy

High levels of microphthalmia transcription factor (MITF) expression have been described in several cell types, including melanocytes, mast cells, and osteoclasts. MITF plays a pivotal role in the regulation of specific genes in these cells. Although its mRNA has been found to be present in relatively high levels in the heart, its cardiac role has never been explored. Here we show that a specific heart isoform of MITF is expressed in cardiomyocytes and can be induced by beta-adrenergic stimulation but not by paired box gene 3 (PAX3), the regulator of the melanocyte MITF isoform. In 2 mouse strains with different MITF mutations, heart weight/body weight ratio was decreased as was the hypertrophic response to beta-adrenergic stimulation. These mice also demonstrated a tendency to sudden death following beta-adrenergic stimulation. Most impressively, 15-month-old MITF-mutated mice had greatly decreased heart weight/body weight ratio, systolic function, and cardiac output. In contrast with normal mice, in the MITF-mutated mice, beta-adrenergic stimulation failed to induce B-type natriuretic peptide (BNP), an important modulator of cardiac hypertrophy, while atrial natriuretic peptide levels and phosphorylated Akt were increased, suggesting a cardiac stress response. In addition, cardiomyocytes cultured with siRNA against MITF showed a substantial decrease in BNP promoter activity. Thus, for what we believe is the first time, we have demonstrated that MITF plays an essential role in beta-adrenergic-induced cardiac hypertrophy.

The Role of Phospholipid Hydroperoxide Glutathione Peroxidase Isoforms in Murine Embryogenesis

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.

Proteome analysis of myocardial tissue following ischemia and reperfusion—Effects of complement inhibition

Myocardial ischemia–reperfusion injury can be related to complement activation with generation of chemotactic mediators, release of cytokines, leukocyte accumulation, and subsequent severe tissue injury. In this regard, activation of transcription factors (i.e., NFκB) and de novo protein synthesis or inflammatory protein degradation seems to play an important role. In the present study, we analyzed the cardiac protein expression following myocardial ischemia (60 min) and reperfusion (180 min) in a rabbit model utilizing two-dimensional electrophoresis and nanoHPLC/ESI-MS/MS for biochemical protein identification. To achieve cardioprotective effects, we used a novel highly selective small molecule C1s inhibitor administered 5 min prior to reperfusion. The reduction of myocardial injury was observed as diminished plasma creatine kinase activity in C1s-INH-248-treated animals (65.2 ± 3 vs. 38.5 ± 3 U/g protein after 3 h of reperfusion, P < 0.05). With proteome analysis we were able to detect 509 ± 21 protein spots on the gels of the 3 groups. A pattern of 480 spots with identical positions was found on every gel of myocardial tissue of sham animals, vehicle and C1s-INH-248-treated animals. We analyzed 11 spots, which were identified by mass spectrometry: Superoxide dismutase, α-crystallin-chain-B, mitochondrial stress protein, Mn SOD, ATP synthase A chain heart isoform, creatine kinase, and troponin T. All of these proteins were significantly decreased in the vehicle group when we compared to sham-treated animals. Treatment with C1s-INH-248 preserved levels of these proteins. Thus, blocking the classical complement pathway with a highly specific and potent synthetic inhibitor of the activated C1 complex archives cardio-protection by altering and preserving different anti-inflammatory and cytoprotective cascades.