Myosin light chain kinase mediates sarcomere organization during cardiac hypertrophy in vitro.

During the development of hypertrophy, cardiac myocytes increase organization of the sarcomere, a highly ordered contractile unit in striated muscle cells. Several hypertrophic agonists, such as angiotensin II, phenylephrine, and endothelin-1, have been shown to promote the sarcomere organization. However, the signaling pathway, which links extracellular stimuli to sarcomere organization, has not been clearly demonstrated. Here, we demonstrate that myosin light chain kinase specifically mediates agonist-induced sarcomere organization during early hypertrophic response. Acute administration of a hypertrophic agonist, phenylephrine, or angiotensin II, causes phosphorylation of myosin light chain 2v both in cultured cardiac myocytes and in the adult heart in vivo. We also show that both sarcomere organization and myosin light chain 2v phosphorylation are dependent on the activation of Ca2+/calmodulin pathway, a known activator of myosin light chain kinase. These results define a new and specific role of myosin light chain kinase in cardiac myocytes, which may provide a rapid adaptive mechanism in response to hypertrophic stimuli.

Effects of neostigmine and physostigmine on the acetylcholine receptor-ionophore complex in frog isolated sympathetic neurones.

1. The effects of neostigmine and physostigmine, reversible carbamate acetylcholinesterase (AChE)-inhibitors, on nicotinic acetylcholine-induced inward currents (IACh) were investigated in enzymatically isolated single sympathetic ganglion cells from the bullfrog. The 'concentration clamp' technique which combines intracellular perfusion with a rapid external solution change under single electrode voltage-clamp conditions was used. 2. Pretreatment with neostigmine and physostigmine did not enhance IACh at any concentrations, suggesting that AChE activity had already disappeared during the enzymatic treatment of the preparation. 3. Both neostigmine and physostigmine inhibited IACh in a dose-dependent manner with IC50 values of 7.0 x 10(-4) M and 1.1 x 10(-4) M, respectively. The blockade by neostigmine was competitive, while that by physostigmine was non-competitive. 4. The inhibition of IACh by neostigmine and physostigmine showed no apparent voltage dependency. 5. Neostigmine did not cause obvious changes of the kinetics of IACh. However, physostigmine reduced both the fast and slow time constants of inactivation of IACh, thus facilitating the rate of inactivation without affecting the activation kinetics of IACh. 6. These results suggest that neostigmine and physostigmine have different direct actions on the ACh receptor-ionophore complex. Neostigmine may act on the ACh-receptor (the binding site of ACh) while physostigmine may interact with the ACh-gated cation channels.

Two cases of dilated cardiomyopathy complicated by left ventricular aneurysm.

This report describes two patients with dilated cardiomyopathy associated with angiographically proven left ventricular aneurysm. There was no apparent history of myocardial infarction and coronary arteries were angiographically normal in both cases.

Inhibition of nicotinic acetylcholine response by atropine in frog isolated sympathetic neurons.

The effect of atropine on nicotinic acetylcholine (ACh) response was studied in frog isolated sympathetic ganglion cells using a 'concentration clamp' technique which combines intra-cellular perfusion with a rapid external solution change within 3 ms. When atropine (more than 6 x 10(-7) M) was simultaneously applied to neurons with ACh (6 x 10(-6) M), the current amplitude was instantaneously reduced in a dose-dependent fashion. Further decreases in the current amplitude were observed in a time-dependent manner during about 20 min after the start of drug-application. Thereafter the current amplitude gradually restored toward the control level in the following 90-120 min in spite of the continuous presence of atropine. However, because d-tubocurarine greatly inhibited the 'restored' current, the last-mentioned is suggested to be also mediated by nicotinic ACh receptor-ionophore complex. Therefore, the inhibitory action of atropine on the peak amplitude of ACh response was examined at 15-20 min after adding the agent. It was dose-dependent but not voltage-dependent. Respective concentrations of atropine and d-tubocurarine causing half the maximum inhibition (IC50) of the peak current evoked by ACh (6 x 10(-6) M) were 1.8 x 10(-5) M and 1.8 x 10(-6) M. Thus, the inhibitory potency was 10 times less than that of d-tubocurarine, an antagonist of nicotinic ACh receptors. The blockade of ACh response by d-tubocurarine was competitive while that by atropine was non-competitive. The current elicited by ACh consisted of two (fast and slow) exponential components plus a steady-state one in the control period.(ABSTRACT TRUNCATED AT 250 WORDS)

Different actions of intracellular free calcium on resting and GABA-gated chloride conductances.

The effects of voltage-dependent Ca2+ current (ICa) on resting and gamma-aminobutyric acid (GABA)-gated Cl- conductances were studied in isolated frog sensory neurons. The amplitude of GABA-gated Cl- current (ICl) was greatly suppressed by a preceding ICa. The GABA dose-response curve was shifted to the right without changing the maximum response by increasing [Ca2+]i. An ICa-activated ICl was observed as an inward tail current on the 'off' phase of ICa. This tail current was easily saturated by increasing the amount of Ca2+ influx.

Some properties of inhibitory action of lidocaine on the Ca2+ current of single isolated frog sensory neurons.

The action of lidocaine on the Ca2+ current (ICa) was studied on single isolated neurons of frog dorsal root ganglia using a 'concentration-clamp' technique which combines intracellular perfusion with a rapid external solution change. Lidocaine decreased the peak amplitude of ICa at a threshold concentration of 10 microM. Higher concentrations gave a dose-dependent increase in inhibition of ICa. Lidocaine also depressed the Na+ current (INa) at a threshold concentration 10 times lower than that for decreasing the amplitude of ICa of neurons isolated from same ganglia. The rate of inhibitory action on ICa was slowed at more negative holding potentials (VH). Lidocaine appears to block both closed and open Ca2+ channels, but acts more profoundly on open channels.

The role of autophagy in the heart.

Autophagy has evolved as a conserving process that uses bulk degradation and recycling of cytoplasmic components, such as long-lived proteins and organelles. In the heart, autophagy is important for the turnover of organelles at low basal levels under normal conditions and it is upregulated in response to stresses such as ischemia/reperfusion and in cardiovascular diseases such as heart failure. Cardiac remodeling involves increased rates of cardiomyocyte cell death and precedes heart failure. The simultaneously occurring multiple features of failing hearts include not only apoptosis and necrosis but also autophagy as well. However, it has been unclear as to whether autophagy is a sign of failed cardiomyocyte repair or is a suicide pathway for failing cardiomyocytes. The functional role of autophagy during ischemia/reperfusion in the heart is complex. It has also been unclear whether autophagy is protective or detrimental in response to ischemia/reperfusion in the heart. In this review, we will summarize the role of autophagy in the heart under both normal conditions and in response to stress.

Caffeine affects four different ionic currents in the bull-frog sympathetic neurone.

1. Ionic mechanisms related to the caffeine-induced current (Icaffeine) were examined in the single isolated sympathetic neurones of the bull-frog. We used the 'concentration-jump' technique in combination with intracellular perfusion and a rapid external solution change, under single-electrode voltage-clamp conditions. 2. Icaffeine was pharmacologically separated into a tetraethylammonium (TEA)-sensitive transient outward current (ITO), a picrotoxin (PTX)-sensitive transient inward current (ITI) and a TEA- and PTX-insensitive sustained inward current (ISI). At low concentrations of caffeine, a sustained outward current (ISO) was observed instead of ISI. 3. All components of Icaffeine were abolished by intracellular perfusion of 30 mM-EGTA. Pre-treatment with A23187 or ryanodine or the simultaneous application of procaine either reduced or abolished all the components of Icaffeine in a dose-dependent manner. The concentration causing 50% inhibition (IC50) was 10(-8) M for A23187 and 2 mM for procaine. 4. The peak response of ITO increased abruptly at caffeine concentrations between 3 and 6 mM followed by saturation above 30 mM. A notch was observed on the rising phase of ITO. 5. The reversal potential (Ecaffeine) of ITO shifted 58 mV for a tenfold change of the extracellular K+ concentration. External application of TEA blocked ITO with an IC50 of 1 mM. ITO was relatively insensitive to apamin, 4-aminopyridine and muscarine. 6. In external solution containing 2 mM-Ca2+, ITO induced by 10 mM-caffeine recovered completely within 3 min from a previous exposure to caffeine. In the absence of extracellular Ca2+, there was little such recovery. A 5 min treatment in a Ca2+-free solution reduced ITO induced by the first application of caffeine by 5%. With a continuous application of 3 mM-caffeine, the amplitude of ITO induced by 10 mM-caffeine reduced in 1 min, and showed a partial recovery in 3 min. The amplitude of ITO increased by increasing the concentration of intracellular Cl-. 7. ITI was activated around the peak of ITO and was rapidly inactivated. ITI was evoked at caffeine concentrations of about 6-10 mM. When the intracellular Cl- concentration was changed, the amplitude of ITI behaved like a Cl- electrode. The Ecaffeine of ITI was close to the Cl- equilibrium potential (ECl). 8. ISI was a 'plateau' response and persisted for over 3 min. ISI was due to a decrease in K+ conductance. In the presence of muscarine (3 x 10(-5) M), ISI was occluded.(ABSTRACT TRUNCATED AT 400 WORDS)

Tyrosine kinases mediation of c-fos expression by cell swelling in cardiac myocytes.

We investigated the signal transduction mechanism initiated by hypotonic cell swelling in cardiac myocytes using c-fos expression as a nuclear marker. Treatment of myocytes with hypotonic culture media rapidly induced c-fos expression, whereas hypertonic stress had no effect on it. Extensive pharmacological studies indicated that only tyrosine kinase inhibitors suppressed the hypotonic swelling-induced c-fos expression; down-regulation of protein kinase C or a Ca2+ chelator (EGTA) had no effect. Hypotonic stress immediately (within 5s) increased tyrosine kinase activity. Thus tyrosine kinase is one of the earliest signaling molecules activated by hypotonic stress and plays an essential role in hypotonic swelling-induced c-fos expression. Rapid activation of tyrosine kinase may be a common early signaling mechanism in response to mechanical stresses that increase cell membrane tension.

The cellular and molecular response of cardiac myocytes to mechanical stress.

External load plays a critical role in determining muscle mass and its phenotype in cardiac myocytes. Cardiac myocytes have the ability to sense mechanical stretch and convert it into intracellular growth signals, which lead to hypertrophy. Mechanical stretch of cardiac myocytes in vitro causes activation of multiple second messenger systems that are very similar to growth factor-induced cell signaling systems. Stretch of neonatal rat cardiac myocytes stimulates a rapid secretion of angiotensin II which, together with other growth factors, mediates stretch-induced hypertrophic responses in vitro. In this review, various cell signaling mechanisms initiated by mechanical stress on cardiac myocytes are summarized with emphasis on potential mechanosensing mechanisms and the relationship between mechanical loading and the cardiac renin-angiotensin system.

Kinetic properties of the caffeine-induced transient outward current in bull-frog sympathetic neurones.

1. The kinetic properties of the caffeine-induced transient outward current (ICaff) of the bull-frog sympathetic neurone were investigated using the extremely rapid concentration-jump technique. By setting the holding potential at the equilibrium potential for Cl- (-50 mV), the involvement of the Ca(2+)-activated Cl- current was suppressed. Using a Na(+)-free (Tris) external solution, the involvement of the Na(+)-dependent sustained outward current was eliminated. The 'M' conductance was also occluded by pre-treatment with muscarine. Under these experimental conditions, ICaff consisted of a TEA-sensitive Ca(2+)-activated K+ current. 2. When the latent period from the application of caffeine until the onset of ICaff (termed the ICaff latency) was measured, 10 mM-caffeine gave a latency of 10.5 +/- 0.7 ms (n = 14, mean +/- S.E.M.) at 22 degrees C. The latency was independent of caffeine concentration between 3 and 30 mM. 3. The ICaff latency was temperature-dependent; it was shortened when the temperature was elevated. 4. Both the time to peak and half-decay time of ICaff were decreased with increasing caffeine concentration. In each cell, these parameters decreased by increasing the amplitude of ICaff. 5. At 22 degrees C, the time to peak and the half-decay time of ICaff elicited by 10 mM-caffeine showed a linear relationship, and this relationship was preserved on either elevating or lowering the temperature. On lowering the temperature (12 degrees C), the time to peak shortened whereas the half-decay time was prolonged. On elevating the temperature (32 degrees C), the time to peak was prolonged whereas the half-decay time was shortened. 6. When EGTA in the intracellular solution was replaced by equimolar BAPTA, the time to peak was prolonged while the half-decay time was shortened. 7. It is concluded that caffeine can activate ICaff, with a time course in the order of milliseconds, and that the kinetics of activation and inactivation of ICaff reflect the time-dependent change in the total amount of intracellular free Ca2+.

Rapamycin selectively inhibits angiotensin II-induced increase in protein synthesis in cardiac myocytes in vitro. Potential role of 70-kD S6 kinase in angiotensin II-induced cardiac hypertrophy.

It has been suggested that phosphorylation of a 40S ribosomal protein, S6, regulates protein synthesis. Two distinct families of S6 kinase have been identified, the rsk-encoded 85- to 92-kD S6 kinase (RSK) and the 70- or 85-kD S6 kinase (p70S6K). We have previously shown that hypertrophic stimuli, such as angiotensin II (Ang II), rapidly activate RSK in cardiac myocytes. However, RSK and p70S6K are regulated by distinct mechanisms, and p70S6K, but not RSK, is the physiological S6 kinase in vivo in other cell types. Using cultured neonatal rat ventricular myocytes, we examined whether Ang II activates p70S6K and investigated the effect of rapamycin, a potent yet indirect inhibitor of p70S6K, on the Ang II-induced hypertrophic response. Immunoblot analyses indicate that cardiac myocytes express the 70- and 85-kD forms of p70s6K. Ang II caused a rapid and sustained activation of p70S6K through the type I Ang II receptor. Rapamycin inhibited Ang II-induced activation of p70S6K in a dose-dependent manner, with an IC50 of 0.14 ng/mL (0.15 nmol/L). Rapamycin did not inhibit Ang II-induced activation of tyrosine kinase, mitogen-activated protein kinase, RSK, and protein kinase C. The effect of rapamycin is unlikely to be mediated by its effect on p34cdc2 and p33cdk2 because Ang II did not activate these cell cycle-dependent kinases in cardiac myocytes. In contrast, a dose-dependent inhibition of p70S6K by rapamycin is very closely correlated with its inhibition of the Ang II-induced increase in protein synthesis. Interestingly, rapamycin did not affect the Ang II-induced activation of specific gene expression, including the immediate-early gene c-fos and fetal type genes, such as atrial natriuretic factor and skeletal alpha-actin. Moreover, rapamycin did not suppress Ang II-induced phenotypic changes at the protein level, such as increased atrial natriuretic factor secretion, expression of beta-myosin heavy chain, and organization of actin into sarcomeric units. These results indicate that p70S6K is activated by Ang II and that a rapamycin-sensitive signaling mechanism, most likely p70S6K, plays an essential role in the Ang II-induced increase in overall protein synthesis but not in Ang II-induced specific phenotypic changes in cardiac myocytes.

The heterotrimeric G q protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes.

p21 ras plays as important role in cell proliferation, transformation and differentiation. Recently, the requirement of p21 ras has been suggested for cellular responses induced by stimulation of heterotrimeric G protein-coupled receptors. However, it remains to be determined how agonists for G protein-coupled receptors activate p21 ras in metazoans. We show here that stimulation of the G q protein-coupled angiotensin II (Ang II) receptor causes activation of p21 ras in cardiac myocytes. The p21 ras activation by Ang II is mediated by an increase in the guanine nucleotide exchange activity, but not by an inhibition of the GTPase-activating protein. Ang II causes rapid tyrosine phosphorylation of Shc and its association with Grb2 and mSos-1, a guanine nucleotide exchange factor of p21 ras. This leads to translocation of mSos-1 to the membrane fraction. Shc associates with the SH3 domain of Fyn whose tyrosine kinase activity is activated by Ang II with a similar time course as that of tyrosine phosphorylation of Shc. Ang II-induced increase in the guanine nucleotide exchange activity was inhibited by a peptide ligand specific to the SH3 domain of the Src family tyrosine kinases. These results suggest that an agonist for a pertussis toxin-insensitive G protein-coupled receptor may initiate the cross-talk with non-receptor-type tyrosine kinases, thereby activating p21 ras using a similar mechanism as receptor tyrosine kinase-induced p21 ras activation.

Molecular characterization of angiotensin II--induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. Critical role of the AT1 receptor subtype.

Increasing evidence suggests that angiotensin II (Ang II) may act as a growth factor for the heart. However, direct effects of Ang II on mammalian cardiac cells (myocytes and nonmyocytes), independent of secondary hemodynamic and neurohumoral effects, have not been well characterized. Therefore, we analyzed the molecular phenotype of cultured cardiac cells from neonatal rats in response to Ang II. In addition, we examined the effects of selective Ang II receptor subtype antagonists in mediating the biological effects of Ang II. In myocyte culture, Ang II caused an increase in protein synthesis without changing the rate of DNA synthesis. In contrast, Ang II induced increases in protein synthesis, DNA synthesis, and cell number in nonmyocyte cultures (mostly cardiac fibroblasts). The Ang II-induced hypertrophic response of myocytes and mitogenic response of fibroblasts were mediated primarily by the AT1 receptor. Ang II caused a rapid induction of many immediate-early genes (c-fos, c-jun, jun B, Egr-1, and c-myc) in myocyte and nonmyocyte cultures. Ang II induced "late" markers for cardiac hypertrophy, skeletal alpha-actin and atrial natriuretic factor expression, within 6 hours in myocytes. Ang II also caused upregulation of the angiotensinogen gene and transforming growth factor-beta 1 gene within 6 hours. Induction of immediate-early genes, late genes, and growth factor genes by Ang II was fully blocked by an AT1 receptor antagonist but not by an AT2 receptor antagonist. These results indicate that: (1) Ang II causes hypertrophy of cardiac myocytes and mitogenesis of cardiac fibroblasts, (2) the phenotypic changes of cardiac cells in response to Ang II in vitro closely mimic those of growth factor response in vitro and of load-induced hypertrophy in vivo, (3) all biological effects of Ang II examined here are mediated primarily by the AT1 receptor subtype, and (4) Ang II may initiate a positive-feedback regulation of cardiac hypertrophic response by inducing the angiotensinogen gene and transforming growth factor-beta 1 gene.

Signal transduction pathways of angiotensin II--induced c-fos gene expression in cardiac myocytes in vitro. Roles of phospholipid-derived second messengers.

Angiotensin II (Ang II) causes a rapid induction of immediate-early genes and hypertrophy in the cardiac myocyte. However, the signaling mechanism of Ang II-induced immediate-early gene expression in cardiac myocytes has not been characterized. Therefore, we examined signal transduction of Ang II in neonatal rat cardiac myocytes, using c-fos gene expression as a model system. Transient transfection of c-fos reporter gene constructs indicated that the serum response element is not only required but also sufficient for Ang II-induced activation of the c-fos promoter. Ang II is known to cause an increase in [Ca2+]i. We found that Ang II also causes a small increase in cAMP in cardiac myocytes. However, the Ca2+/cAMP response element of the c-fos gene was not sufficient to confer Ang II responsiveness to the c-fos promoter, and inhibitors of protein kinase A had no effects on Ang II-induced c-fos expression. On the other hand, chelating intracellular Ca2+ with BAPTA-AM inhibited Ang II-induced c-fos expression in a dose-dependent manner, suggesting that Ca2+ is required for Ang II-induced signaling. Measurements of phospholipid-derived second messengers revealed that Ang II increased production of inositol trisphosphate, diacylglycerol, phosphatidic acid, and arachidonic acids, resulting in a sustained increase in protein kinase C activity. This and other evidence suggest that Ang II activates phospholipase C, phospholipase D, and possibly phospholipase A2. All of these second-messenger systems are activated through the AT1 receptor. Pharmacological inhibition of phospholipase C or downregulation of protein kinase C significantly suppressed Ang II-induced c-fos expression. In conclusion, Ang II activates multiple phospholipid-derived second-messenger systems via the AT1 receptor in cardiac myocytes. Among these second-messenger systems, phospholipase C and protein kinase C seem essential for Ang II-induced c-fos gene expression, whereas Ca2+ may play a permissive role. Finally, the "Ang II response element" of the c-fos gene maps to the protein kinase C-dependent portion of the serum response element.

Mechanotransduction in stretch-induced hypertrophy of cardiac myocytes.

Mechanical loading of cardiac muscles causes rapid activation of a number of immediate-early (IE) genes and hypertrophy. However, little is known as to how muscle cells sense mechanical load and regulate gene expression. We examined roles of several putative mechanotransducers in stretch-induced hypertrophy of cardiac myocytes grown on a deformable silicone sheet. Using the patch-clamp technique, we found a single class of stretch-activated cation channels which was completely and reversibly blocked by gadolinium. The inhibition of this channel by gadolinium did not affect either stretch-induced expression of the IE genes or hypertrophy. Neither disruption of microtubules with colchicine nor that of actin microfilaments by cytochalasin D prevented the stretch-induced IE gene expression. Arresting contractile activity by tetrodotoxin did not affect the stretch-induced IE gene expression or hypertrophy. These results suggest that stretch-activated cation channels, microtubules, microfilaments, and contractile activity are not the mechanotransducers. Preliminary results suggest that cell stretch may cause a release of a growth factor(s), which in turn initiates a cascade of hypertrophic response of cardiac myocytes.

Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism.

It is well known that external load plays a critical role in determining cardiac muscle mass and its phenotype, but little is known as to how mechanical load is transduced into intracellular signals regulating gene expression. To address this question we analyzed the 'mechano-transcription' coupling process using an in vitro model of load-induced cardiac hypertrophy, in which a stretch of rat cardiac myocytes, grown on a deformable substrate, causes a rapid induction of immediate-early genes followed by growth (hypertrophic) response. We report here that cell stretch rapidly activates a plethora of second messenger pathways, including tyrosine kinases, p21ras, mitogen-activated protein (MAP) kinases, S6 kinases (pp90RSK), protein kinase C, phospholipase C, phospholipase D, and probably the phospholipase A2 and P450 pathways. In contrast, the cAMP pathway is not activated significantly by stretch. The signals generated by these second messengers appear to converge into activation of the p67SRF-p62TCF complex via the serum response element, causing induction of c-fos. The stretch response may involve an autocrine or paracrine mechanism, because stretch-conditioned medium, when transferred to non-stretched myocytes, mimicked the effect of stretch. These results indicate that mechanical load causes rapid activation of multiple second messenger systems, which may in turn initiate a cascade of hypertrophic response of cardiac myocytes.

Angiotensin II and serum differentially regulate expression of cyclins, activity of cyclin-dependent kinases, and phosphorylation of retinoblastoma gene product in neonatal cardiac myocytes.

The hypertrophic response in cardiac myocytes and the mitogenic response in other cell types share various early cellular responses. However, how the subsequent cell growth response, such as cell cycle machinery, is regulated in cardiac hypertrophy is not understood. Using cultured neonatal rat cardiac myocytes, we examined the effect of angiotensin II (Ang II), a hypertrophic stimulus, on mRNA and protein expression of cyclins and cyclin-dependent protein kinases (cdks), activity of cdks, and phosphorylation of retinoblastoma gene product (pRb). The effect of FCS, a stimulus that was previously reported to initiate both protein and DNA synthesis in cardiac myocytes, was also examined for comparison. Ang II activated cdk4 and caused phosphorylation of pRb, peaking at 12 hours, but subsequently downregulated cyclin D1, D3, and A expression and cdk2 activity. FCS increased the expression of G1-S cyclins, caused activation of cdk4, cdk2, and cdc2, and strongly phosphorylated pRb but failed to significantly stimulate DNA synthesis in neonatal cardiac myocytes. These results suggest that Ang II transiently activates but subsequently downregulates cell cycle regulators. Induction of G1 and G1-S cyclins and activation of cdks by FCS are not sufficient to drive cardiac myocytes into S phase. The functional role of pRb phosphorylation by Ang II and serum stimulation and, by inference, the subsequent liberation of E2F in terminally differentiated myocytes remain to be elucidated.