Monoacylglycerol lipase activity in cardiac myocytes

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100,000 X g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100,000 X g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 microM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 microM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA greater than free fatty acids greater than fatty acyl carnitine.     

Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes

In cardiac myocytes, sustained (3 min) intracellular acidosis activates the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway and, through this pathway, increases sarcolemmal NHE (Na+/H+ exchanger) activity [Haworth, McCann, Snabaitis, Roberts and Avkiran (2003) J. Biol. Chem. 278, 31676-31684]. In the present study, we aimed to determine the time-dependence, pH-dependence and upstream signalling mechanisms of acidosis-induced ERK1/2 activation in ARVM (adult rat ventricular myocytes). Cultured ARVM were subjected to intracellular acidosis for up to 20 min by exposure to NH4Cl, followed by washout with a bicarbonate-free Tyrode solution containing the NHE1 inhibitor cariporide. After the desired duration of intracellular acidosis, the phosphorylation status of ERK1/2 and its downstream effector p90(RSK) (90 kDa ribosomal S6 kinase) were determined by Western blotting. This revealed a time-dependent transient phosphorylation of both ERK1/2 and p90(RSK) by intracellular acidosis (intracellular pH approximately 6.6), with maximum activation occurring at 3 min and a return to basal levels by 20 min. When the degree of intracellular acidosis was varied from approximately 6.8 to approximately 6.5, maximum ERK1/2 phosphorylation was observed at an intracellular pH of 6.64. Inhibition of MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK kinase 1/2) by pre-treatment of ARVM with U0126 or adenoviral expression of dominant-negative D208A-MEK1 protein prevented the phosphorylation of ERK1/2 by sustained intracellular acidosis, as did inhibition of Raf-1 with GW 5074 or ZM 336372. Interference with Ras signalling by the adenoviral expression of dominant-negative N17-Ras protein or with FPT III (farnesyl protein transferase inhibitor III) also prevented acidosis-induced ERK1/2 phosphorylation, whereas inhibiting G-protein signalling [by adenoviral expression of RGS4 or Lsc, the RGS domain of p115 RhoGEF (guanine nucleotide-exchange factor)] or protein kinase C (with bisindolylmaleimide I) had no effect. Our data show that, in ARVM, sustained intracellular acidosis activates ERK1/2 through proximal activation of the classical Ras/Raf/MEK pathway.     

Cytoskeletal prestress regulates nuclear shape and stiffness in cardiac myocytes

Mechanical stresses on the myocyte nucleus have been associated with several diseases and potentially transduce mechanical stimuli into cellular responses. Although a number of physical links between the nuclear envelope and cytoplasmic filaments have been identified, previous studies have focused on the mechanical properties of individual components of the nucleus, such as the nuclear envelope and lamin network. The mechanical interaction between the cytoskeleton and chromatin on nuclear deformability remains elusive. Here, we investigated how cytoskeletal and chromatin structures influence nuclear mechanics in cardiac myocytes. Rapid decondensation of chromatin and rupture of the nuclear membrane caused a sudden expansion of DNA, a consequence of prestress exerted on the nucleus. To characterize the prestress exerted on the nucleus, we measured the shape and the stiffness of isolated nuclei and nuclei in living myocytes during disruption of cytoskeletal, myofibrillar, and chromatin structure. We found that the nucleus in myocytes is subject to both tensional and compressional prestress and its deformability is determined by a balance of those opposing forces. By developing a computational model of the prestressed nucleus, we showed that cytoskeletal and chromatin prestresses create vulnerability in the nuclear envelope. Our studies suggest the cytoskeletal–nuclear–chromatin interconnectivity may play an important role in mechanics of myocyte contraction and in the development of laminopathies by lamin mutations.     

RhoA GTPase regulates L-type Ca2+ currents in cardiac myocytes

Regulation of ionic channels plays a pivotal role in controlling cardiac function. Here we show that the Rho family of small G proteins regulates L-type Ca2+ currents in ventricular cardiomyocytes. Ventricular myocytes isolated from transgenic (TG) mice that overexpress the specific GDP dissociation inhibitor Rho GDI-alpha exhibited significantly decreased basal L-type Ca2+ current density (approximately 40%) compared with myocytes from nontransgenic (NTG) mice. The Ca2+ channel agonist BAY K 8644 and the beta-adrenergic agonist isoproterenol increased Ca2+ currents in both NTG and TG myocytes to a similar maximal level, and no changes in mRNA or protein levels were observed in the Ca2+ channel alpha1-subunits. These results suggest that the channel activity but not the expression level was altered in TG myocytes. In addition, the densities of inward rectifier and transient outward K+ currents were unchanged in TG myocytes. The amplitudes and rates of basal twitches and Ca2+ transients were also similar between the two groups. When the protein was delivered directly into adult ventricular myocytes via TAT-mediated protein transduction, Rho GDI-alpha significantly decreased Ca2+ current density, which supports the idea that the defective Ca2+ channel activity in TG myocytes was a primary effect of the transgene. In addition, expression of a dominant-negative RhoA but not a dominant-negative Rac-1 or Cdc42 also significantly decreased Ca2+ current density, which indicates that inhibition of Ca2+ channel activity by overexpression of Rho GDI-alpha is mediated by inhibition of RhoA. This study points to the L-type Ca2+ channel activity as a novel downstream target of the RhoA signaling pathway.     

Testosterone regulates mRNA levels of calcium regulatory proteins in cardiac myocytes.

Gender-related differences in cardiac function have been described in the literature, but whether the presence of sex hormones is responsible for these differences remains unclear. This study was designed to determine whether testosterone regulates the gene expression of calcium regulatory proteins in rat heart, thus playing a role in gender-related differences in cardiac performance. Ventricular myocytes were isolated from two-day-old rats and treated with testosterone at varying duration; the levels of gene expression for the androgen receptor (AR) and major calcium regulatory proteins were determined by quantitative real-time PCR. Testosterone (1 microM) treatment induced a maximum increase in beta1-adrenergic receptor and L-type calcium channel mRNA levels following an eight hour exposure. Six hours testosterone treatment stimulated a 300-fold increase in androgen receptor message abundance, and Na/Ca exchanger mRNA levels reached a maximum level following twenty-four hour testosterone treatment. Taken together, these data provide the first evidence that testosterone regulates gene expression of the major calcium regulatory proteins in isolated ventricular myocytes, and may thus play a role in the gender-related differences observed in cardiac performance.     

c-Src regulates the interaction between connexin-43 and ZO-1 in cardiac myocytes

Connexin-43 is known to interact directly with ZO-1 in cardiac myocytes, but little is known about the role of ZO-1 in connexin-43 function. In cardiac myocytes, constitutively active c-Src inhibited endogenous interaction between connexin-43 and ZO-1 by binding to connexin-43. In HEK293 cells, by contrast, a connexin-43 mutant lacking the Src phosphorylation site (Tyr265) interacted with ZO-1 despite cotransfection of a constitutively active c-Src. Moreover, in vitro binding assays using recombinant proteins synthesized from regions of connexin-43 and ZO-1 showed that the tyrosine-phosphorylated C terminus of connexin-43 interacts with the c-Src SH2 domain in parallel with the loss of its interaction with ZO-1. Cell surface biotinylation revealed that, by phosphorylating Tyr265, constitutively active c-Src reduces total and cell surface connexin-43 down to the levels seen in cells expressing a mutant connexin-43 lacking the ZO-1 binding domain. Finally, electrophysiological analysis showed that both the tyrosine phosphorylation site and the ZO-1-binding domain of connexin-43 were involved in the regulation of gap junctional function. We therefore conclude that c-Src regulates the interaction between connexin-43 and ZO-1 through tyrosine phosphorylation and through the binding of its SH2 domain to connexin-43.     

Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes

The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in several cardiac pathologies including hypertrophy and failure. In this review we describe the role that the cytoskeleton plays in modulating both the electrical activity (through ion channels and exchangers) and mechanical (or contractile) activity of the adult heart. We focus on the 3 components of the cytoskeleton, actin microfilaments, microtubules, and desmin filaments. The limited visual data available suggest that the subsarcolemmal actin cytoskeleton is sparse in the adult myocyte. Selective disruption of cytoskeletal actin by pharmacological tools has yet to be verified in the adult cell, yet evidence exists for modulation of several ionic currents, including I(CaL), I(Na), I(KATP), I(SAC) by actin microfilaments. Microtubules exist as a dense network throughout the adult cardiac cell, and their structure, architecture, kinetics and pharmacological manipulation are well described. Both polymerised and free tubulin are functionally significant. Microtubule proliferation reduces contraction by impeding sarcomeric motion; modulation of sarcoplasmic reticulum Ca(2+) release may also be involved in this effect. The lack of effect of microtubule disruption on cardiac contractility in adult myocytes, and the concentration-dependent modulation of the rate of contraction by the disruptor nocodazole in neonatal myocytes, support the existence of functionally distinct microtubule populations. We address the controversy regarding the stimulation of the beta-adrenergic signalling pathway by free tubulin. Work with mice lacking desmin has demonstrated the importance of intermediate filaments to normal cardiac function, but the precise role that desmin plays in the electrical and mechanical activity of cardiac muscle has yet to be determined.     

Calcium not strain regulates localization of alpha-myosin heavy chain mRNA in oriented cardiac myocytes

Our aim was to test a hypothesis that localization of the alpha-myosin heavy chain (alpha-MyHC) mRNA in oriented neonatal rat cardiomyocytes is regulated either by calcium, or by mechanical strain, or by both. Myocytes, grown on collagen aligned on stretchable silicone membranes, were elongated and had an increased length to width ratio (L/W) compared with randomly oriented myocytes grown on conventional substrata. Oriented cells were stretched by 10% in the longitudinal direction, in the transverse direction or passively unloaded for 6 h. As expected, shape changes followed these mechanical deformations. In situ hybridization was used to determine the localization of alpha-MyHC mRNA by quantitative analysis of optical density under various mechanical perturbations in myocytes that were either spontaneously beating or treated with verapamil (10 mM) to block influx of calcium. Unstretched, longitudinally stretched, and cells stretched transversely all had mRNA dispersed to their extremities. Verapamil treatment resulted in a perinuclear pattern of mRNA under all three mechanical perturbations. Additionally, mRNA distribution was examined in myocytes that were passively unloaded in the presence and absence of verapamil. Unloading myocytes with intact calcium cycling does not result in a perinuclear accumulation of mRNA. These data suggest that calcium is essential for alpha-MyHC mRNA distribution throughout the cell whereas stretch and alignment affect myocyte shape but have little effect on mRNA localization.     

5'-Nucleotidase activity of isolated mature rat cardiac myocytes

Specific location of 5'-nucleotidase in the heart has been uncertain, some authors citing evidence for an exclusively non-myocyte location, while other data point to the existence of cytoplasmic and membrane-bound fractions. Single myocytes isolated from mature rat heart, and free of endothelial or interstitial cells, have been used to establish that muscle cells of the myocardium are rich in 5'-nucleotidase, exhibiting activity sufficient to account for the total myocardial content of this enzyme. All 5'-nucleotidase is accessible to extracellular AMP. Inhibitors of 5'-nucleotidase and adenosine transport have been used to establish that only the adenosine component of adenine nucleotides is taken up by myocytes, but hydrolysis of AMP by 5'-nucleotidase does not commit the adenosine formed to transport across the sarcolemmal membrane. Myocytes also have ecto-phosphatases which hydrolyse ADP and ATP.     

Calcineurin/NFAT signaling in osteoblasts regulates bone mass

Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.     

Hdac2 regulates the cardiac hypertrophic response by modulating Gsk3 beta activity

In the adult heart, a variety of stresses induce re-expression of a fetal gene program in association with myocyte hypertrophy and heart failure. Here we show that histone deacetylase-2 (Hdac2) regulates expression of many fetal cardiac isoforms. Hdac2 deficiency or chemical histone deacetylase (HDAC) inhibition prevented the re-expression of fetal genes and attenuated cardiac hypertrophy in hearts exposed to hypertrophic stimuli. Resistance to hypertrophy was associated with increased expression of the gene encoding inositol polyphosphate-5-phosphatase f (Inpp5f) resulting in constitutive activation of glycogen synthase kinase 3beta (Gsk3beta) via inactivation of thymoma viral proto-oncogene (Akt) and 3-phosphoinositide-dependent protein kinase-1 (Pdk1). In contrast, Hdac2 transgenic mice had augmented hypertrophy associated with inactivated Gsk3beta. Chemical inhibition of activated Gsk3beta allowed Hdac2-deficient adults to become sensitive to hypertrophic stimulation. These results suggest that Hdac2 is an important molecular target of HDAC inhibitors in the heart and that Hdac2 and Gsk3beta are components of a regulatory pathway providing an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure.     

5′-Nucleotidase activity of isolated mature rat cardiac myocytes

Specific location of 5′-nucleotidase in the heart has been uncertain, some authors citing evidence for an exclusively non-myocyte location, while other data point to the existence of cytoplasmic and membrane-bound fractions. Single myocytes isolated from mature rat heart, and free of endothelial or interstitial cells, have been used to establish that muscle cells of the myocardium are rich in 5′-nucleotidase, exhibiting activity sufficient to account for the total myocardial content of this enzyme. All 5′-nucleotidase is accessible to extracellular AMP. Inhibitors of 5′-nucleotidase and adenosine transport have been used to establish that only the adenosine component of adenine nucleotides is taken up by myocytes, but hydrolysis of AMP by 5′-nucleotidase does not commit the adenosine formed to transport across the sarcolemmal membrane. Myocytes also have ecto-phosphatases which hydrolyse ADP and ATP.     

Calcium binding to cardiac myocytes protected from proteolytic enzyme activity

Excitation-contraction coupling in cardiac muscle is dependent on extracellular calcium and calcium bound to the surface of the myocardial cell. In this study, we examined the physical characteristics of calcium binding to adult guinea pig ventricular myocytes disaggregated mechanically in oxygenated tissue culture medium containing a proteinase inhibitor (aprotinin), and separated from cellular debris by Cytodex beads. Cells prepared in this manner excluded Trypan blue and showed no evidence of spontaneous contraction or contracture. Scatchard plots of calcium binding determined by continuous flow equilibrium dialysis revealed a high-affinity, low-capacity pool, Ka = 65 X 10(3) M-1 and Bt = 1.3 nmol X mg-1 and a low-affinity, high-capacity pool, Ka = 141 M-1 and Bt = 138 nmol X mg-1. The low-affinity pool was not detectable after lanthanum, trypsin or collagenase treatment or in cells prepared without aprotinin in the isolation medium. Both neuraminidase and phospholipase C reduced Bt of the low-affinity pool by one half, but only neuraminidase affected the affinity constant of this pool. Ka was increased to 516.7 M-1, similar to the apparent affinity constant for calcium binding estimated from dP/dtmax measured at several extracellular calcium concentrations (470 M-1). The results suggest that calcium bound to sarcolemmal phospholipids represents the superficial calcium involved in excitation-contraction coupling in the heart.