Sarcomeric Alpha-actinin Research Articles

FoxO1 Activation Promotes Proapoptotic Gene Expression In Differentiated Skeletal Muscle In Vitro

The winged-helix transcription factor FoxO1 (forkhead box, class O) plays an important role in the regulation of cell cycle, cell growth and cell survival. Recent findings have demonstrated attenuated sarcomeric alpha actinin expression and loss of myotube formation in differentiated skeletal muscle following FoxO1 activation in vitro. The mechanisms responsible for mediating the changes in myotube structure, however, remain to be elucidated. PURPOSE To determine the gene expression changes in differentiated skeletal muscle following FoxO1 activation in skeletal myotubes in vitro. METHODS FoxO1-estrogen receptor (FoxO1-ER) fusion proteins that are activated by treatment with 4-hydroxytamoxifen (4-OHT) were stably transfected in C2C12 skeletal myoblasts using the pBABE retroviral system. FoxO1-ER transfected or control myoblasts (empty construct) were differentiated into myotubes in low serum and maintained for 4 days. On the 4th day following serum deprivation, total RNA was isolated from skeletal myotubes following treatment with either vehicle (control; 0.1% ethanol final concentration) or 4-OHT (500 nM) for 12 hours. 15 μg of labeled cRNA per sample was hybridized to mouse gene chips, washed and gene expression analyzed performed. All conditions were performed in triplicate and pooled for subsequent analysis. Semi-quantitative RT-PCR was performed for conformational analysis on several genes of interest demonstrating induction or repression following FoxO1 activation. Amplification products were normalized to the housekeeping gene GAPDH. RESULTS Gene expression analysis revealed that a total of 55 genes demonstrated significant changes (+/- 2 fold log ratio change; 20 induction, 35 repression) following 12h treatment with 4-OHT, including several genes associated with apoptosis, cytoskeletal maintenance, proteolysis, and cell size regulation. RT-PCR analysis on follistatin (1.6 fold induction), growth arrest and DNA-damage-inducible 45⊓ (2.6 fold induction), ubiquitin specific protease 2 (2.2 fold induction), and metalloproteinase-13 (undetectable transcript levels following 4-OHT treatment) confirmed the findings of the gene expression analysis. CONCLUSION These initial findings indicate that transcriptional activation of FoxO1 may impact skeletal muscle growth and/or repair by regulating genes responsible for maintaining myotube organization and survival. Supported by NIH grants T32 HL07692-12 (TJM), AR45671 (KAE), and DK-41430 (TGU) and the Department of Veterans Affairs Merit Review Program (TGU).

Cholesterol depletion by methyl-?-cyclodextrin enhances myoblast fusion and induces the formation of myotubes with disorganized nuclei

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-beta-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric alpha-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and beta-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.

Dynamics of obscurin localization during differentiation and remodeling of cardiac myocytes: obscurin as an integrator of myofibrillar structure.

Obscurin is a newly identified giant muscle protein whose functions remain to be elucidated. In this study we used high-resolution confocal microscopy to examine the dynamics of obscurin localization in cultures of rat cardiac myocytes during the assembly and disassembly of myofibrils. Double immunolabeling of neonatal and adult rat cells for obscurin and sarcomeric alpha-actinin, the major protein of Z-lines, demonstrated that, during myofibrillogenesis, obscurin is intensely incorporated into M-band areas of A-bands and, to a lesser extent, in Z-lines of newly formed sarcomeres. Presarcomeric structural precursors of myofibrils were intensely immunopositive for alpha-actinin and, unlike mature myofibrils, weakly immunopositive or immunonegative for obscurin. This indicates that most of the obscurin assembles in developing myofibrils after abundant incorporation of alpha-actinin and that massive integration of obscurin occurs at more advanced stages of sarcomere assembly. Immunoreactivity for obscurin in the middle of A-bands and in Z-lines of sarcomeres bridged the gaps between individual bundles of newly formed myofibrils, suggesting that this protein appears to be directly involved in their primary lateral connection and registered alignment into larger clusters. Close sarcomeric localization of obscurin and titin suggests that they may interact during myofibril assembly. Interestingly, the laterally aligned striated pattern of obscurin formed at a stage when desmin, traditionally considered as a molecular linker responsible for the lateral binding and stabilization of myofibrils at the Z-bands, was still diffusely localized. During the disassembly of the contractile system in adult myocytes, disappearance of the cross-striated pattern of obscurin preceded the disorganization of registered alignment and intense breakdown of myofibrils. The cross-striated pattern of desmin typical of terminally differentiated myocytes disappeared before or simultaneously with obscurin. During redifferentiation, as in neonatal myocytes, sarcomeric incorporation of obscurin closely followed that of alpha-actinin and occurred earlier than the striated arrangement of desmin intermediate filaments. The presence of obscurin in the Z-lines and its later assembly into the A/M-bands indicate that it may serve to stabilize and align sarcomeric structure when myosin filaments are incorporated. Our data suggest that obscurin, interacting with other muscle proteins and possibly with the sarcoplasmic reticulum, may have a role as a flexible structural integrator of myofibrils during assembly and adaptive remodeling of the contractile apparatus.

Inhibitors arrest myofibrillogenesis in skeletal muscle cells at early stages of assembly

A three-step model for myofibrillogenesis has been proposed for the formation of myofibrils [Rhee et al., 1994: Cell Motil. Cytoskeleton 28:1-24; Sanger et al., 2002: Adv. Exp. Med. 481:89-105]: premyofibril to nascent myofibril to mature myofibril. We have found two chemically related inhibitors that will arrest development at both the first and second step. Cultured quail embryonic skeletal myoblasts were treated with ethyl methane sulfonate (EMS) or 2-aminoethyl-methanesulfonate (MTSEA+). When the myoblasts fused in the presence of either of these compounds, myosheets rather than myotubes formed. Treated cells were fixed and immunostained against multiple proteins commonly found in muscle cells. Protein expression and localization throughout the myosheet were similar to that of developing myotube tips. Cells treated with high concentrations of EMS (10 mM) stained for non-muscle myosin II, sarcomeric alpha-actinin, and tropomyosin. No zeugmatin (Z-band region of titin) or muscle myosin II antibody staining was detected in fibers in this treatment group. These fibers are comparable to premyofibrils in control myotubes. At lower concentrations of EMS (7.5 to 5 mM), fibers that formed stained for muscle myosin II and titin as well as for non-muscle myosin IIB, sarcomeric alpha-actinin, and tropomyosin. Muscle myosin II was in an unbanded pattern. These fibers are comparable to nascent myofibrils observed during normal myofibrillogenesis. Similar effects to those obtained by treating cells with EMS were obtained when we treated cultured cells with MTSEA+ (5 mM) and stained them with sarcomeric alpha-actinin. MTSEA+ is chemically related to EMS, and is a well-known inhibitor of ryanodine receptors in skeletal muscle cells. Some abnormalities such as nemaline-like rods and other protein aggregates also appear within the myosheet during EMS and MTSEA+ treatment. Removal of these two inhibitors of myofibrillogenesis allows the premyofibrils and nascent myofibrils to form mature myofibrils.

Inhibition of phenylephrine-induced cardiac hypertrophy by docosahexaenoic acid.

Many of the cardiovascular benefits of fish oil result from the antiarrhythmic actions of the n-3 polyunsaturated lipids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The beneficial effects of DHA/EPA in patients with coronary artery disease and myocardial infarction may also result from modulation of the myocardial hypertrophic response. Hypertrophy was assessed in neonatal cardiomyocytes exposed to phenylephrine (PE) by measuring cell surface area, total protein synthesis ((14)C leucine incorporation), and the organization of sarcomeric alpha-actinin and by monitoring expression of atrial natriuretic factor (ANF). We report that PE induced a twofold increase in cell surface area and protein synthesis in cardiomyocytes. The hypertrophied cardiomyocytes also exhibited increased expression of ANF in perinuclear regions and organization of sarcomeric alpha-actinin into classical z-bands. Treatment of cardiomyocytes with 5 microM DHA effectively prevented PE-induced hypertrophy as shown by inhibition of surface area expansion and protein synthesis, inhibition of ANF expression, and prevention of alpha-actinin organization into z-bands. DHA treatment prevented PE-induced activation of Ras and Raf-1 kinase. The upstream inhibition of Ras --> Raf-1 effectively prevented translocation and nuclear localization of phosphorylated extracellularly regulated kinase 1 and 2 (Erk1/2). These effects consequently led to inhibition of nuclear translocation, and hence, activation of the downstream signaling enzyme p90 ribosomal S6 kinase (p90(rsk)). These results indicate that PE-induced cardiac hypertrophy can be minimized by DHA. Our results suggest that inhibition of Ras --> Raf-1 --> Erk1/2 --> p90(rsk) --> hypertrophy is one possible pathway by which DHA can inhibit cardiac hypertrophy. In vivo studies are needed to confirm these in vitro effects of DHA.

Anticonvulsant valproic acid inhibits cardiomyocyte differentiation of embryonic stem cells by increasing intracellular levels of reactive oxygen species.

The anticonvulsant valproic acid (VPA) exerts teratogenic properties and has been demonstrated to cause neural tube defects and malformations of the heart. The effect of VPA on the differentiation of cardiomyocytes from pluripotent murine embryonic stem cells (ES cells) was investigated. Embryoid bodies derived from ES cells were treated with different concentrations of VPA and the differentiation of cardiomyocytes was monitored by immunohistochemical staining for sarcomeric alpha-actinin. Cytotoxicity was evaluated by the use of the dead cell stain SYTOX green. Intracellular levels of reactive oxygen species (ROS) within the tissue were evaluated by the use of the redox-sensitive dye dichlorodihydrofluorescein diacetate (H2DCFDA). VPA retarded the growth of ES cell-derived embryoid bodies but did not exert cytotoxic effects. The compound dose-dependently inhibited the development of spontaneously beating clusters of cardiomyocytes within embryoid bodies grown from ES cells and reduced the extension of beating areas of cardiac cells. Furthermore, VPA significantly increased ROS levels, indicating that VPA altered the intracellular redox balance. To investigate whether the inhibition of cardiomyocyte differentiation by VPA was owing to increased ROS overwhelming the intracellular antioxidative defense, the compound was coadministered with the free radical scavenger vitamin E. This treatment significantly restored cardiomyogenic differentiation, indicating that VPA inhibits cardiomyogenesis of ES cells by increasing intracellular ROS levels.

3,5,3'-Triiodothyronine deprivation affects phenotype and intracellular [Ca2+]i of human cardiomyocytes in culture.

A decrease in plasma T3 concentration is a frequent finding in patients with heart failure. However, the role of this 'low T3 syndrome' on disease evolution has never been clarified. As phenotypic and functional cardiomyocyte impairments are alterations that correlate with the failing myocardium, we studied the long-term effects of T3 deprivation on human cardiomyocyte structure and calcium handling. Atrial cardiomyocytes and myocardial tissue were cultured with or without 3 nM T3. Microscopical examination of structural features was followed by analysis of alpha-sarcomeric actinin and sarcoplasmic reticulum calcium ATP-ase (SERCA-2) content. Calcium handling was studied by [Ca2+](i) imaging. When stimulated with cyclopiazonic acid, a SERCA-2 inhibitor, T3-deprived cardiomyocytes showed significantly faster (P=0.03) and more transient (P=0.04) increases in [Ca(2+)](i) than T3-supplemented cells. Moreover, in the T3-free cultures a significantly lower number of cells (P=0.003) responded to caffeine, a typical activator of sarcoplasmic reticulum Ca(2+)-release channel. T3-deprived cardiomyocytes also presented altered morphology with larger dimensions than T3-supplemented cells (P < 0.0001). Additionally, in T3-deprived samples alpha-sarcomeric actinin and SERCA-2 protein levels were reduced to 65.6 +/- 3% (P < 0.0001) and 74.1 +/- 4% (P=0.005), respectively, when compared with the T3-supplemented group. Our data show that human cardiomyocyte calcium handling and phenotype are strongly influenced by T3 suggesting important implications of the 'low T3 syndrome' on the progression of heart failure.

Formation of sarcomeres in developing myotubes: role of mechanical stretch and contractile activation.

In a series of experiments, cultured myotubes were exposed to passive stretch or pharmacological agents that block contractile activation. Under these experimental conditions, the formation of Z lines and A bands (morphological structures, resulting from the specific structural alignment of sarcomeric proteins, necessary for contraction) was assessed by immunofluorescence. The addition of an antagonist of the voltage-gated Na(+) channels [tetrodotoxin (TTX)] for 2 days in developing rat myotube cultures led to a nearly total absence of Z lines and A bands. When contractile activation was allowed to resume for 2 days, the Z lines and A bands reappeared in a significant way. The appearance of Z lines or A bands could not be inhibited nor facilitated by the application of a uniaxial passive stretch. Electrical stimulation of the cultures increased sarcomere assembly significantly. Antagonists of L-type Ca(2+) channels (verapamil, nifedipine) combined with electrical stimulation led to the absence of Z lines and A bands to the same degree as the TTX treatment. Western blot analysis did not show a major change in the amount of sarcomeric alpha-actinin nor a shift in myosin heavy chain phenotype as a result of a 2-day passive stretch or TTX treatment. Results of experiments suggest that temporal Ca(2+) transients play an important factor in the assembly and maintenance of sarcomeric structures during muscle fiber development.

Heart Protection Using a New Approach - Antisense to Angiotensin Converting Enzyme Mrna

P200 Introduction. Angiotensin converting enzyme (ACE), plays a major role in many cardiovascular pathophysiological conditions, such as hypertension, heart failure and myocardial ischemia. Currently used ACE inhibitors have some adverse effects and non-compliance problem. Therefore, our lab has been developing an antisense approach to inhibit the renin-angiotensin system at the gene expression level. The present study had three aims: 1. Design antisense oligodeoxynucleotide (AS-ODN) directed at ACE mRNA 2. Check tissue distribution of AS after iv injection 3. Test AS for heart protection in ischemia- reperfusion injury Methods. Epifluorescent and confocal laser-scanning microscopy, immunohistochemistry, Western blotting. Animals : mice, males, 20 g and Sprague-Dawley rats, males, 200-250 g. AS 5’ -ATTTCGTGGTGGG-3’ , inverted control (INV) 5’ -GGGTGGTGCTTTA-3’, phosphorothioates mixed with liposomes and injected iv 0.04 mg/mouse and 0.2 mg/rat. Captopril dose was 5 mg/kg. Isolated perfused heart function recordings : coronary perfusion pressure (CPP), left ventricular end-diastolic (LVEDP) and systolic pressures (LVSP). Results. Based on the rat ACE cDNA sequence, the AS was designed as 13-mer directed to the second zinc-binding domain (2998-3010 bp). Tissue distribution of phosphorothioated, fluorescein isothiocyanate (FITC)-labeled AS, 4h after iv injection, showed its presence in the lung, spleen, liver and heart, but absence in the brain. Immunostaining of the parallel heart slices with anti sarcomeric alpha-actinin antibody indicated AS localization within cardiomyocytes. ACE AS injected iv to the rats 24 h before excising the heart, which was then subjected to 25 min ischemia and 30 min reperfusion, preserved heart function. Increase in CPP and LVEDP during reperfusion was attenuated by AS as compared to INV or saline treatment (n=8 in each group, p

Heart Protection Using a New Approach - Antisense to Angiotensin Converting Enzyme Mrna

P200 Introduction. Angiotensin converting enzyme (ACE), plays a major role in many cardiovascular pathophysiological conditions, such as hypertension, heart failure and myocardial ischemia. Currently used ACE inhibitors have some adverse effects and non-compliance problem. Therefore, our lab has been developing an antisense approach to inhibit the renin-angiotensin system at the gene expression level. The present study had three aims: 1. Design antisense oligodeoxynucleotide (AS-ODN) directed at ACE mRNA 2. Check tissue distribution of AS after iv injection 3. Test AS for heart protection in ischemia- reperfusion injury Methods. Epifluorescent and confocal laser-scanning microscopy, immunohistochemistry, Western blotting. Animals : mice, males, 20 g and Sprague-Dawley rats, males, 200-250 g. AS 5’ -ATTTCGTGGTGGG-3’ , inverted control (INV) 5’ -GGGTGGTGCTTTA-3’, phosphorothioates mixed with liposomes and injected iv 0.04 mg/mouse and 0.2 mg/rat. Captopril dose was 5 mg/kg. Isolated perfused heart function recordings : coronary perfusion pressure (CPP), left ventricular end-diastolic (LVEDP) and systolic pressures (LVSP). Results. Based on the rat ACE cDNA sequence, the AS was designed as 13-mer directed to the second zinc-binding domain (2998-3010 bp). Tissue distribution of phosphorothioated, fluorescein isothiocyanate (FITC)-labeled AS, 4h after iv injection, showed its presence in the lung, spleen, liver and heart, but absence in the brain. Immunostaining of the parallel heart slices with anti sarcomeric alpha-actinin antibody indicated AS localization within cardiomyocytes. ACE AS injected iv to the rats 24 h before excising the heart, which was then subjected to 25 min ischemia and 30 min reperfusion, preserved heart function. Increase in CPP and LVEDP during reperfusion was attenuated by AS as compared to INV or saline treatment (n=8 in each group, p&lt;0.05). Moreover, ACE protein was not upregulated, as it was in the captopril-treated group. Conclusion. ACE AS is taken up by the heart tissue and provides cardioprotection by effectively preventing myocardial dysfunction caused by ischemia-reperfusion injury.

Tropomodulin assembles early in myofibrillogenesis in chick skeletal muscle: evidence that thin filaments rearrange to form striated myofibrils.

Actin filament lengths in muscle and nonmuscle cells are believed to depend on the regulated activity of capping proteins at both the fast growing (barbed) and slow growing (pointed) filament ends. In striated muscle, the pointed end capping protein, tropomodulin, has been shown to maintain the lengths of thin filaments in mature myofibrils. To determine whether tropomodulin might also be involved in thin filament assembly, we investigated the assembly of tropomodulin into myofibrils during differentiation of primary cultures of chick skeletal muscle cells. Our results show that tropomodulin is expressed early in differentiation and is associated with the earliest premyofibrils which contain overlapping and misaligned actin filaments. In addition, tropomodulin can be found in actin filament bundles at the distal tips of growing myotubes, where sarcomeric alpha-actinin is not always detected, suggesting that tropomodulin caps actin filament pointed ends even before the filaments are cross-linked into Z bodies by alpha-actinin. Tropomodulin staining exhibits an irregular punctate pattern along the length of premyofibrils that demonstrate a smooth phalloidin staining pattern for F-actin. Strikingly, the tropomodulin dots often appear to be located between the closely spaced, dot-like Z bodies that are stained for (&agr;)-actinin. Thus, in the earliest premyofibrils, the pointed ends of the thin filaments are clustered and partially aligned with respect to the Z bodies (the location of the barbed filament ends). At later stages of differentiation, the tropomodulin dots become aligned into regular periodic striations concurrently with the appearance of striated phalloidin staining for F-actin and alignment of Z bodies into Z lines. Tropomodulin, together with the barbed end capping protein, CapZ, may function from the earliest stages of myofibrillogenesis to restrict the lengths of newly assembled thin filaments by capping their ends; thus, transitions from nonstriated to striated myofibrils in skeletal muscle are likely due principally to filament rearrangements rather than to filament polymerization or depolymerization. Rearrangements of actin filaments capped at their pointed and barbed ends may be a general mechanism by which cells restructure their actin cytoskeletal networks during cell growth and differentiation.

Localization and quantitation of cardiac annexins II, V, and VI in hypertensive guinea pigs.

Annexins are characterized by Ca2+-dependent binding to phospholipids. Annexin II mainly participates in cell-cell adhesion and signal transduction, whereas annexins V and VI also seem to regulate intracellular calcium cycling. Their abundance and localization were determined in left ventricle (LV) and right ventricle (RV) from hypertensive guinea pigs, during the transition from compensatory hypertrophy to heart failure. Immunoblot analysis of annexins II, V, and VI revealed an increased accumulation (2.6-, 1.45-, and 2.3-fold, respectively) in LV from hypertensive guinea pigs and no modification in RV. Immunofluorescent labeling of annexins II, V, and VI; of Na+-K+-ATPase; and of sarcomeric alpha-actinin showed that in control LV and RV, 1) annexin II is present in nonmuscle cells; 2) annexins V and VI are mainly observed in the sarcolemma and intercalated disks of myocytes; 3) annexins II, V, and VI strongly label endothelial cells and adventitia of coronary arteries; and 4) annexin VI is present in the media. At the onset of heart failure, the most striking changes are the increased protein accumulation in LV and the very strong labeling of annexins II, V, and VI in interstitial tissue, suggesting a role in fibrosis development and cardiac remodeling.

Hyperplasia and hypertrophy of chicken cardiac myocytes during posthatching development.

For characterization of the growth pattern of cardiac myocytes during posthatching development, cardiac myocytes were enzymatically isolated from the ventricles of 1-, 15-, 29-, and 42-day-old chickens for measurement of myocyte nucleation, length, width, volume, and number, and for immunolabeling of cytoskeletal proteins. Ventricular myocyte number increased 156% from day 1 to day 42. Average cell volume increased more than 400%, and myocytes lengthened 125%, but cell width only increased 53% during this period. All myocytes were mononucleated at day 1. At day 15, 18% of myocytes became binucleated with < 1% of myocytes containing more than two nuclei. Interestingly, binucleated myocytes were able to divide with two nuclei going through mitosis at the same time. As demonstrated by staining with tubulin and alpha-actinin antibodies, two mitotic spindles and two cleavage furrows were formed in dividing binucleated myocytes. At day 42, binucleated myocytes increased to 44% with 11% of myocytes containing more than two nuclei. Sarcomeric alpha-actinin was partially disassembled in prometaphase and was reorganized into regular Z lines of sarcomeres in telophase. Desmin was disassembled in prophase and was reassembled during late telophase. These results suggest that chicken myocytes undergo hypertrophy and continue to proliferate during posthatching maturation, although it is currently believed that myocytes of all vertebrates withdraw from the cell cycle shortly after birth. We provide direct evidence for the first time of in vivo myocyte division in 6-wk-old chicken hearts.

Different compartments of sarcoplasmic reticulum participate in the excitation-contraction coupling process in human atrial myocytes.

The excitation-contraction coupling process of human atrial myocytes was studied in voltage-clamped myocytes isolated from right atrial appendages obtained during cardiac surgery. Intracellular Ca2+ transients (Cai transients) were monitored with 0.1 mmol/L indo 1 added to the internal dialyzing solution. Ryanodine receptors (RyRs) and sarcomeric alpha-actinin were stained with specific antibodies and visualized using plane and confocal microscopy. L-Type Ca2+ current (Ica) elicited a prolonged Cai transient, with an initial rapidly activating phase (slope 1, 23.6 +/- 1.2 s-1) followed by a slowly activating phase (slope 2, 5.8 +/- 0.4 s-1; P < .001 versus slope 1), resulting in a dome-shaped Cai transient. Ryanodine (100 mumol/L) inhibited 79 +/- 6% of the Cai transient, indicating that it was due essentially to sarcoplasmic reticulum Ca2+ release. During step depolarizations, maximal activation of the Cai transient or tail current (Itail) (in cells dialyzed with Ca2+ buffer-free internal solution) preceded that of Ica and did not follow its voltage dependence (n = 12). Test pulses lasting from 5 to 150 milliseconds elicited a similar time course of both Cai transient and Itail (n = 5). In a given cell, the two components of the Cai transient could be dissociated by altering the intracellular Ca2+ load, by increasing the stimulation rate from 0.1 to 1 Hz, or by varying the amplitude of Ica. Immunostaining of atrial sections and isolated myocytes showed that a large number of RyRs were located not only in a subsarcolemmal position but also deeper inside the cell, in a regularly spaced transverse band pattern at the level of Z lines. Together, our results indicate that, in human atrial myocytes, Ica only partially controls the activation of RyRs, with the prolonged and dome-shaped Cai transient of these cells probably reflecting the activation of RyRs not coupled to L-type Ca2+ channels.

Unanticipated temporal and spatial effects of sarcomeric alpha-actinin peptides expressed in PtK2 cells.

To understand the multiple roles of alpha-actinin in the assembly of (1) Z bands in muscle, and (2) a variety of cytoskeletal structures in non-muscle cells, 4 sarcomeric alpha-actinin derived cDNAs tagged with a MYC epitope were constructed. The constructs were: (1) full-length (FL/MYC); (2) minus EF-hands (-EF/MYC); (3) actin-binding site (+A/MYC); and (4) minus actin-binding site (-A/MYC). These four cDNAs were individually transfected into PtK2 cells. The exogenous sarcomeric alpha-actinin (s-alpha-actinin/MYC) was followed with labeled anti-MYC, the endogenous non-sarcomeric alpha-actinin (non-s-alpha-actinin) with labeled anti-non-s-alpha-actinin. The salient findings were: (1) the selective intracellular localizations of each expressed MYC-tagged peptide differed one from the other; (2) their respective localizations in the 10-24-h post-transfection (p.t.) period differed from their localizations in the 48-72-h p.t. period; (3) each MYC-positive peptide was cytotoxic, but each in a distinctive way; and (4) while the selective targeting of FL/MYC to dense bodies, adhesion plaques, adherens junctions, and ruffled membranes was consistent with binding studies in cell-free systems, the incorporation of the mutated peptides, particularly +A/MYC and -A/MYC was not. Changes in localization over time and the distinctive cytopathologies probably reflect domain-specific targeting. They also suggest unexpected cooperative involvement of multiple domains of alpha-actinin with specific receptors in distal cytoskeletal structures. To date, such qualitative in vivo interactions have not been described either in in vitro binding studies, or in short-term experiments involving localization and/or fate of microinjected labeled molecules into living cells.

A sarcomeric alpha-actinin truncated at the carboxyl end induces the breakdown of stress fibers in PtK2 cells and the formation of nemaline-like bodies and breakdown of myofibrils in myotubes.

In many nonmuscle cells, nonsarcomeric alpha-actinin is distributed in the dense bodies of stress fibers, adhesion plaques, and adherens junctions. In striated muscle, a sarcomeric isoform of alpha-actinin (s-alpha-actinin) is found in the Z-bands of myofibrils and subsarcolemmal adhesion plaques. To understand the role(s) of the alpha-actinin isoforms in the assembly and maintenance of such cytoskeletal structures, full-length or truncated s-alpha-actinin cDNAs were expressed in PtK2 cells and in primary skeletal myogenic cells. We found the following. (i) In transfected PtK2 cells the truncated s-alpha-actinin was rapidly incorporated into preexisting dense bodies, adhesion plaques, and adherens junctions. With time these structures collapsed, and the affected cells detached from the substrate. (ii) In myotubes the truncated s-alpha-actinin was incorporated into nascent Z-bands. Many of these progressively hypertrophied, forming nemaline-like bodies. With time the affected myofibrils fragmented, and the myotubes detached from the substrate. (iii) In both cell types the truncated s-alpha-actinin was significantly more disruptive of the cytoskeletal structures than the full-length molecule. (iv) Pools of "over-expressed" full-length or truncated protein did not self-aggregate into homogeneous, amorphous complexes; rather the exogenous proteins selectively colocalized with the same cohort of cytoskeletal proteins with which the endogenous alpha-actinin normally associates. The similarity among the hypertrophied Z-bands in transfected myotubes, the nemaline bodies in patients with nemaline myopathies, and the streaming Z-bands seen in various muscle pathologies raises the possibility that the genetically determined nemaline bodies and the pathologically induced Z-band alterations may reflect primary and/or post-translational modifications of s-alpha-actinin.

Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils.

Cultured cardiac myocytes were stained with antibodies to sarcomeric alpha-actinin, troponin-I, alpha-actin, myosin heavy chain (MHC), titin, myomesin, C-protein, and vinculin. Attention was focused on the distribution of these proteins with respect to nonstriated myofibrils (NSMFs) and striated myofibrils (SMFs). In NSMFs, alpha-actinin is found as longitudinally aligned, irregular approximately 0.3-microns aggregates. Such aggregates are associated with alpha-actin, troponin-I, and titin. These I-Z-I-like complexes are also found as ectopic patches outside the domain of myofibrils in close apposition to the ventral surface of the cell. MHC is found outside of SMFs in the form of discrete fibrils. The temporal-spatial distribution and accumulation of the MHC-fibrils with respect to the I-Z-I-like complexes varies greatly along the length of the NSMFs. There are numerous instances of I-Z-I-like complexes without associated MHC-fibrils, and also cases of MHC-fibrils located many microns from I-Z-I-like complexes. The transition between the terminal approximately 1.7-microns sarcomere of any given SMF and its distal NSMF-tip is abrupt and is marked by a characteristic narrow alpha-actinin Z-band and vinculin positive adhesion plaque. A titin antibody T20, which localizes to an epitope at the Z-band in SMFs, precisely costains the 0.3-microns alpha-actinin aggregates in ectopic patches and NSMFs. Another titin antibody T1, which in SMFs localizes to an epitope at the A-I junction, typically does not stain ectopic patches and NSMFs. Where detectable, the T1-positive material is adjacent to rather than part of the 0.3-microns alpha-actinin aggregates. Myomesin and C-protein are found only in their characteristic sarcomeric locations (even in just perceptible SMFs). These A-band-associated proteins appear to be absent in ectopic patches and NSMFs.

Differential response of myofibrillar and cytoskeletal proteins in cells treated with phorbol myristate acetate.

Muscle-specific and nonmuscle contractile protein isoforms responded in opposite ways to 12-o-tetradecanoyl phorbol-13-acetate (TPA). Loss of Z band density was observed in day-4-5 cultured chick myotubes after 2 h in the phorbol ester, TPA. By 5-10 h, most I-Z-I complexes were selectively deleted from the myofibril, although the A bands remained intact and longitudinally aligned. The deletion of I-Z-I complexes was inversely related to the appearance of numerous cortical, alpha-actinin containing bodies (CABs), transitory structures approximately 3.0 microns in diameter. Each CAB consisted of a filamentous core that costained with antibodies to alpha-actin and sarcomeric alpha-actinin. In turn each CAB was encaged by a discontinuous rim that costained with antibodies to vinculin and talin. Vimentin and desmin intermediate filaments and most cell organelles were excluded from the membrane-free CABs. These curious bodies disappeared over the next 10 h so that in 30-h myosacs all alpha-actin and sarcomeric alpha-actinin structures had been eliminated. On the other hand vinculin and talin adhesion plaques remained prominent even in 72-h myosacs. Disruption of the A bands was first initiated after 15-20 h in TPA (e.g., 15-20-h myosacs). Thick filaments of apparently normal length and structure were progressively released from A segments, and by 40 h all A bands had been broken down into enormous numbers of randomly dispersed, but still intact single thick filaments. This breakdown correlated with the formation of amorphous cytoplasmic aggregates which invariably colocalized antibodies to myosin heavy chain, MLC 1-3, myomesin, and C protein. Complete elimination of all immunoreactive thick filament proteins required 60-72 h of TPA exposure. The elimination of the thick filament-associated proteins did not involve the participation of vinculin or talin. In contrast to its effects on myofibrils, TPA did not induce the disassembly of the contractile proteins in stress fibers and microfilaments either in myosacs or in fibroblastic cells. Similarly, TPA, which rapidly induces the translocation of vinculin and talin to ectopic sites in many types of immortalized cells, had no gross effect on the adhesion plaques of myosacs, primary fibroblastic cells, or presumptive myoblasts. Clearly, the response to TPA of contractile protein and some cytoskeletal isoforms not only varies among phenotypes, but even within the domains of a given myotube the myofibrils respond one way, the stress fibers/microfilaments another.