Overexpression of chemokine-like factor 2 promotes the proliferation and survival of C2C12 skeletal muscle cells.
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Chemokine-like factor 1 (CKLF1) is a novel cytokine first cloned from U937 cells. It contains different splicing forms and has chemotactic effects on a wide spectrum of cells both in vitro and in vivo; it can also stimulate the regeneration of skeletal muscle cells in vivo, but the mechanism remains unclear. To probe the myogenesis function of CKLF2, which is the largest isoform of CKLFs, C2C12 murine myoblasts were stably transfected with human CKLF2 eukaryotic expression vector. Compared with control vector transfected C2C12 cells, CKLF2 overexpression causes accelerated myoblast proliferation as determined by cell counting and [(3)H]TdR incorporation assays. In addition, CKLF2 overexpression also promotes cell differentiation, which was determined by higher expression levels of myogenin, creatine kinase, myosin and the accelerated myoblast fusion. Further analysis also indicates that CKLF2 could activate the transcription activity of the bHLH/MyoD and MEF2 families. Finally, DNA synthesis and myotube formation could also be promoted by growing C2C12 cells in conditioned media from CKLF2-transfected cells. These findings strongly suggest a role for human CKLF2 in regulation of skeletal muscle myogenesis. (+info)
Molecular mechanics of mouse cardiac myosin isoforms.
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Two myosin isoforms are expressed in myocardium, alphaalpha-homodimers (V(1)) and betabeta-homodimers (V(3)). V(1) exhibits higher velocities and myofibrillar ATPase activities compared with V(3). We also observed this for cardiac myosin from normal (V(1)) and propylthiouracil-treated (V(3)) mice. Actin velocity in a motility assay (V(actin)) over V(1) myosin was twice that of V(3) as was the myofibrillar ATPase. Myosin's average force (F(avg)) was similar for V(1) and V(3). Comparing V(actin) and F(avg) across species for both V(1) and V(3), our laboratory showed previously (VanBuren P, Harris DE, Alpert NR, and Warshaw DM. Circ Res 77: 439-444, 1995) that mouse V(1) has greater V(actin) and F(avg) compared with rabbit V(1). Mouse V(3) V(actin) was twice that of rabbit V(actin). To understand myosin's molecular structure and function, we compared alpha- and beta-cardiac myosin sequences from rodents and rabbits. The rabbit alpha- and beta-cardiac myosin differed by eight and four amino acids, respectively, compared with rodents. These residues are localized to both the motor domain and the rod. These differences in sequence and mechanical performance may be an evolutionary attempt to match a myosin's mechanical behavior to the heart's power requirements. (+info)
Prevention of hypertrophy by overexpression of Kv4.2 in cultured neonatal cardiomyocytes.
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BACKGROUND: Prolonged action potentials (APs) and decreased transient outward K+ currents (I(to)) are consistent findings in hypertrophic myocardium. However, the connection of these changes with cardiac hypertrophy is unknown. The present study investigated the effects of changes in I(to) and the associated alterations in AP on myocyte hypertrophy induced by phenylephrine. METHODS AND RESULTS: Chronic incubation of cultured neonatal ventricular rat myocytes (NVRMs) with phenylephrine (PE) reduced I(to) density and prolonged AP duration, leading to a 2-fold increase in the net Ca2+ influx per beat and a 1.4-fold increase in Ca2+-transient amplitude. PE treatment of chronically paced (2-Hz) NVRM also induced increases in cell size, protein/DNA ratio, atrial natriuretic factor mRNA expression, as well as beta/alpha myosin mRNA ratio. These hypertrophic changes were associated with a 2.4-fold increase in activation of nuclear factor of activated T-cells (NFAT), indicating increased activity of the Ca2+-dependent phosphatase calcineurin. Overexpression of Kv4.2 channels using adenovirus prevented the AP duration prolongation as well as the increases in Ca2+ influx and Ca2+-transient amplitude induced by PE. Kv4.2 overexpression also prohibited the PE-induced increases in cell size, protein/DNA ratio, atrial natriuretic factor expression, beta/alpha myosin mRNA ratio, and NFAT activation. CONCLUSIONS: Our results demonstrate that PE-mediated hypertrophy in NRVMs seems to require I(to) reductions and AP prolongation associated with increased Ca2+ influx and Ca2+ transients as well as calcineurin activation. The clinical implications of these studies and the possible involvement of other signaling pathways are discussed. (+info)
Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins.
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OBJECTIVE: The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure. METHODS: Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. RESULTS: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness. CONCLUSIONS: The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI. (+info)
The effect of myosin light chain 2 dephosphorylation on Ca2+ -sensitivity of force is enhanced in failing human hearts.
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OBJECTIVE: Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1+/-2.0%) compared to donor (31.9+/-4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. METHODS: Cooperative effects in isometric force and rate of force redevelopment (K(tr)) were studied in single Triton-skinned human cardiomyocytes at various [Ca(2+)] before and after protein phosphatase-1 (PP-1) incubation. RESULTS: Maximum force and K(tr) values did not differ between failing and donor hearts, but Ca(2+)-sensitivity of force (pCa(50)) was significantly higher in failing myocardium (Deltap Ca(50)=0.17). K(tr) decreased with decreasing [Ca(2+)], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa(50) to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute K(tr) values was found after PP-1 in failing and donor myocytes, while the shape of the K(tr)-Ca(2+) relationships remained unaltered. CONCLUSIONS: Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca(2+)-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca(2+)-sensitivity and impaired Ca(2+)-handling on diastolic function in human heart failure. (+info)
Increased phosphorylation of myosin light chain associated with slow-to-fast transition in rat soleus.
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In striated muscles myosin light chain (MLC)2 phosphorylation regulates calcium sensitivity and mediates sarcomere organization. Little is known about the changes in MLC2 phosphorylation in relation to skeletal muscle plasticity. We studied changes in MLC2 phosphorylation in rats receiving three treatment conditions causing slow-to-fast transitions: 1) atrophy induced by 14 days of hindlimb suspension (HS), 2) hypertrophy induced by 14 days of clenbuterol administration (CB), and 3) 14 days of combined treatment (CB-HS). Three variants of the slow (MLC2s) and two variants of the fast MLC2 (MLC2f) isoform were separated with two-dimensional electrophoresis and identified with monoclonal and polyclonal antibodies specific for MLC2; their relative proportions were densitometrically quantified. In control soleus muscle MLC2s predominated over MLC2f (91.4 +/- 3.9% vs. 8.5 +/- 3.9%) and was separated into two spots, the less acidic spot being 73.5 +/- 4.3% of the total. All treatments caused a decrease of the less acidic unphosphorylated spot of MLC2s (CB: 64.1 +/- 5.6%, HS: 62.4 +/- 6.8%, CB-HS: 56.4 +/- 4.4%), the appearance of a third more acidic variant of MLC2s (representing 3.9-5.9% of total MLC2s), an increase of MLC2f (CB: 30.9 +/- 3.1%, HS: 23.9 +/- 3.3%, CB-HS: 25.3 +/- 3.9%), and the phosphorylation of a large fraction of MLC2f (CB: 30.4 +/- 6.7%, HS: 28.7 +/- 6.5%, CB-HS: 21.8 +/- 2.1%). Treatment with alkaline phosphatase or with protein phosphatase 1 (PP1) removed the most acidic spots of both MLC2f and MLC2s. We conclude that in rat skeletal muscles an increase of MLC2 phosphorylation is associated with the slow-to-fast transition regardless of whether hypertrophy or atrophy develops. (+info)
Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube.
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OBJECTIVE: After formation of the linear heart tube a chamber-specific program of gene expression becomes active that underlies the formation of the chamber myocardium. To assess whether this program is recapitulated in in vitro differentiated embryonic stem cells, we performed qualitative and quantitative analyses of cardiogenesis in vivo and in vitro. METHODS: Gene expression profiles were made by in situ hybridisation and real-time PCR and electrophysiological profiles by patch clamp analyses of cardiomyocytes derived from time series of differentiating HM1 mouse embryonic stem cells and from embryonic and adult mouse hearts. RESULTS: In embryoid bodies the in situ patterns of expression of alpha-myosin heavy chain, myosin light chain 2a and sarcoendoplasmic reticulum calcium ATPase 2a were similar to that of the heart muscle-specific marker gene cardiac troponin I. Myosin light chain 2v was expressed in part of the cardiac troponin I-expressing area, indicating heterogeneity within the cardiac cell population. Atrial natriuretic factor expression, indicative of the chamber-type program, could only very occasionally be detected by in situ hybridisation. Quantitative reverse transcriptase PCR showed that all cardiac genes, most notably atrial natriuretic factor, were expressed at relatively low levels, similar to those in embryonic hearts at embryonic day 8.75-9. Analysis of the electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes showed an increase of the upstroke velocity and a shorter duration of the action potential during prolonged differentiation in vitro. When embryonic mouse heart compartments of embryonic day 12.5 were used as a reference, the electrophysiological characteristics of a substantial part of the embryonic stem cell-derived cardiomyocytes were most reminiscent to those observed in the embryonic outflow tract. CONCLUSION: Together, these data suggest that most cardiomyocytes acquired by differentiation of embryonic stem cells maintain a phenotype reminiscent of that of the cardiomyocytes of the primary heart tube, and hardly any myocytes develop a chamber myocardial phenotype. (+info)
The human adult cardiomyocyte phenotype.
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AIM: Determination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology. METHODS: Atrial and ventricular cardiomyocytes were obtained from patients undergoing cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured with or without serum. The relative cell attachment promoting efficiency of several reagents was evaluated and compared. Morphological changes during long-term culture were assessed with phase contrast microscopy, morphometric analysis and immunocytochemistry or RT-PCR of sarcomeric markers including alpha-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin. RESULTS: The isolation method produced viable rod-shaped atrial (16.6+/-6.0%, mean+/-S.E.; n=5) and ventricular cells (22.4+/-8.0%, mean+/-S.E.; n=5) in addition to significant numbers of apoptotic and necrotic cells. Cell dedifferentiation was characterized by the loss of sarcomeric structure, condensation and extrusion of sarcomeric proteins. Cells cultured with low serum recovered and assumed a flattened, spread form with two distinct morphologies apparent. Type I cells were large, had extensive sarcolemmal spreading, with stress fibers and nascent myofibrils, whilst type II cells appeared smaller, with more mature myofibril organisation and focal adhesions. CONCLUSION: Characterization of the redifferentiation capabilities of cultured adult cardiac myocytes in culture, provides an important system for comparing cardiomyocytes differentiating from human stem cells and provides the basis for an in vitro transplantation model to study interaction and communication between primary adult and stem cell-derived cardiomyocytes. (+info)