Inhibition of cAMP-Dependent PKA Activates ß2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates ß2-adrenergic receptor (ß2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates ß2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 µM; zint-ß2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 µM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 µM GW5074), phospholipase C (PLC; 0.5 µM edelfosine), PKC (4 µM chelerythrine) or IP3 receptor (IP3R) signaling (2 µM 2-APB) significantly inhibited zint-ß2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-ß2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-ß2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-ßgal. In +LMN myocytes, zint-ß2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-ßgal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 µM thapsigargin failed to inhibit zint-ß2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-ß-cyclodextrin inhibited zint-ß2-AR stimulation of ICa,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows ß2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of ß-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

Global comparison of phosphoproteins in human and rodent hearts: implications for translational studies of myosin light chain and troponin phosphorylations.

Cardiac remodeling and failure are regulated by a myriad of cardiac protein phosphorylations. In the present study, cardiac phosphoprotein patterns were examined in rodent and human hearts Left ventricular tissue samples were obtained from human systolic failing (n = 5) and control (n = 5) hearts and from two rat models of hypertensive heart failure, i.e., spontaneously hypertensive heart failure and Dahl salt-sensitive rats and corresponding controls. Phosphoproteins were separated by 2D-DIGE with Cydye staining, phosphoprotein patterns were analyzed using pixel intensity in rectified images. Specific phosphoproteins which were different in human versus rodent hearts were identified by MALDI-TOF/TOF Mass Spectrometry. Targeted pair-wise analyses showed differences (p < 0.05) in 26 % of the pixels, which included pixels containing phosphorylated troponin T, myosin light chain, peroxiredoxin, and haptoglobin. These results show differences in rodent versus human cardiac remodeling which will influence the translation rodent studies to humans in this area.

FRNK overexpression limits the depth and frequency of vascular smooth muscle cell invasion in a three-dimensional fibrin matrix.

Pathological vascular smooth muscle cell (VSMC) behavior after vascular interventions such as angioplasty or bypass is initiated within the 3D environment of the vessel media. Here VSMCs proliferate, invade the surrounding matrix, migrate adluminally, and deposit substantial amounts of matrix, leading to myointimal hyperplasia and decreased blood flow to critical organs and tissue. Since focal adhesion kinase (FAK) mediates many of the VSMC responses to these pathologic events, it provides a reasonable pharmacologic target to limit this invasive VSMC behavior and to better understand the cellular pathophysiology of this disease. Here we quantified the effectiveness of disabling FAK in VSMCs with its dominant-negative inhibitor, FAK-related nonkinase (FRNK), in a clinically relevant 3D assay. We found that FRNK overexpression decreased VSMC invasion (both the length and frequency) in this matrix. These effects were demonstrated in the presence and absence of chemical mitotic inhibition, suggesting that FAK's effect on cellular matrix invasion, migration, and proliferation utilize separate and/or redundant signaling cascades. Mechanistically, FAK inhibition decreased its localization to focal adhesions which led to a significant decrease in FAK autophosphorylation and the phosphorylation of the serine/threonine kinase, AKT. Together these findings suggest that disruption of FAK signaling may provide a pharmaceutical tool that limits pathological VSMC cell behavior.

Laminin enhances beta(2)-adrenergic receptor stimulation of L-type Ca(2+) current via cytosolic phospholipase A(2) signalling in cat atrial myocytes.

We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN), decreases adenylate cyclase (AC)/cAMP and increases beta(2)-adrenergic receptor (AR) stimulation of L-type Ca(2+) current (I(Ca,L)). This study therefore sought to determine whether LMN enhances beta(2)-AR signalling via a cAMP-independent mechanism, i.e. cytosolic phospholipase A(2) (cPLA(2)) signalling. Studies were performed on acutely isolated atrial myocytes plated on uncoated coverslips (LMN) or coverslips coated with LMN (+LMN). As previously reported, 0.1 microm zinterol (zint-beta(2)-AR) stimulation of I(Ca,L) was larger in +LMN than LMN myocytes. In +LMN myocytes, zint-beta(2)-AR stimulation of I(Ca,L) was inhibited by inhibition of cPLA(2) by arachidonyltrifluoromethyl ketone (AACOCF(3); 10 microm), inhibition of G(i) by pertussis toxin and chelation of intracellular Ca(2+) by 10 microm BAPTA-AM. In contrast to zinterol, stimulation of I(Ca,L) by fenoterol (fen-beta(2)-AR), a beta(2)-AR agonist that acts exclusively via G(s) signalling, was smaller in +LMN than LMN myocytes. Arachidonic acid (AA; 5 microm) stimulated I(Ca,L) to a similar extent in LMN and +LMN myocytes. Inhibition of cAMP-dependent protein kinase A (cAMP/PKA) by either 5 mum H89 or 1 microm KT5720 in LMN myocytes mimicked the effects of +LMN myocytes to enhance zint-beta(2)-AR stimulation of I(Ca,L), which was blocked by 10 microm AACOCF(3). In contrast, H89 inhibited fen-beta(2)-AR stimulation of I(Ca,L), which was unchanged by AACOCF(3). Inhibition of ERK1/2 by 1 microm U0126 inhibited zint-beta(2)-AR stimulation of I(Ca,L) in +LMN myocytes and LMN myocytes in which cAMP/PKA was inhibited by KT5720. In LMN myocytes, cytochalasin D prevented inhibition of cAMP/PKA from enhancing zint-beta(2)-AR stimulation of I(Ca,L). We conclude that LMN enhances zint-beta(2)-AR stimulation of I(Ca,L) via G(i)/ERK1/2/cPLA(2)/AA signalling which is activated by concomitant inhibition of cAMP/PKA signalling and dependent on the actin cytoskeleton. These findings provide new insight into the cellular mechanisms by which the extracellular matrix can remodel beta(2)-AR signalling in atrial muscle.

Laminin acts via focal adhesion kinase/phosphatidylinositol-3' kinase/protein kinase B to down-regulate beta1-adrenergic receptor signalling in cat atrial myocytes.

We previously reported that short-term (2 h) plating of cat atrial myocytes on the extracellular matrix protein, laminin (LMN) decreases adenylate cyclase activity and beta(1)-adrenergic receptor (beta(1)-AR) stimulation of L-type Ca(2+) current (I(Ca,L)). The present study sought to determine whether LMN-mediated down-regulation of beta(1) signalling is due to down-regulation of adenylate cyclase and to gain insight into the signalling mechanisms responsible. beta(1)-AR stimulation was achieved by 0.01 microm isoproterenol (isoprenaline) plus 0.1 microm ICI 118551, a selective beta(2)-AR antagonist. Atrial myocytes were plated for at least 2 h on uncoated cover-slips (-LMN) or cover-slips coated with LMN (+LMN). As previously reported, beta(1)-AR stimulation of I(Ca,L) was significantly smaller in +LMN compared to -LMN atrial myocytes. In -LMN myocytes, 10 microm LY294002 (LY), a specific inhibitor of PI-(3)K, had no effect on beta(1)-AR stimulation of I(Ca,L). In +LMN myocytes, however, LY significantly increased beta(1)-AR stimulation of I(Ca,L). Western blots revealed that compared with -LMN myocytes, +LMN myocytes showed a significant increase in Akt phosphorylation at Ser-473, which was prevented by LY. In another approach, +LMN myocytes were infected (multiplicity of infection (MOI), 100; 24 h) with replication-defective adenoviruses (Adv) expressing dominant-negative inhibitors of focal adhesion kinase (FAK) (Adv-FRNK or Adv-Y397F-FAK) or Akt (Adv-dnAkt). Compared with control cells infected with Adv-beta-galactosidase, cells infected with Adv-FRNK, Adv-Y397F-FAK or Adv-dnAkt each exhibited a significantly greater beta(1)-AR stimulation of I(Ca,L). In -LMN myocytes LY had no effect on forskolin (FSK)-stimulated I(Ca,L). However, in +LMN myocytes LY significantly increased FSK-stimulated I(Ca,L). Similar results were obtained in +LMN atrial myocytes infected with Adv-FRNK. We conclude that LMN binding to beta(1)-integrin receptors acts via FAK/PI-(3)K/Akt to inhibit adenylate cyclase activity and thereby down-regulates beta(1)-AR-mediated stimulation of I(Ca,L). These findings provide new insight into the cellular mechanisms by which the extracellular matrix can modulate atrial beta-AR signalling.

Signalling mechanisms in contraction-mediated stimulation of intracellular NO production in cat ventricular myocytes.

In this study we sought to determine whether contractile activity has a role as a signalling mechanism in the activation of intracellular nitric oxide (NO(i)) production induced by electrical stimulation of cat ventricular myocytes. Field stimulation (FS) of single ventricular myocytes elicited frequency-dependent increases in NO(i) that were blocked by the calmodulin (CaM) inhibitor 10 microM W-7 and partially inhibited by the phosphatidylinositol 3'-kinase (PI-(3)K) inhibitor 10 microMm LY294002. Increasing extracellular [Ca(2+)] caused a concentration-dependent increase in FS-induced NO(i) that was partially inhibited by LY294002. The negative inotropic agents BDM (5 mm) or blebbistatin (10 microM) decreased cell shortening and NO(i) production without concomitant changes in L-type Ca(2+) current (I(Ca,L)) or [Ca(2+)](i) transients. The positive inotropic agents EMD 57033 or CGP 48506 (1 microM) increased cell shortening and NO(i) production without concomitant changes in I(Ca,L) or [Ca(2+)](i) transients. FS-induced NO(i) production was decreased in myocytes infected (100 multiplicity of viral infection (MOI); 24 h) with a replication-deficient adenovirus expressing a dominant-negative mutant of protein kinase B (Akt) compared with cells infected with a control adenovirus expressing beta-galactosidase. FS-induced NO(i) was partially inhibited by either endothelial (eNOS) or neuronal nitric oxide synthase (nNOS) inhibitors and completely blocked by simultaneous exposure to both. FS-induced [Ca(2+)](i) transients were increased by the nNOS inhibitor nNOS-I (0.24 microM), decreased by the eNOS inhibitor L-NIO (1 microM) and unchanged by exposure to both inhibitors. We conclude that in cat ventricular myocytes, FS-induced NO(i) production requires both Ca(2+)-dependent CaM signalling and Ca(2+)-independent PI-(3)K-Akt signalling activated by contractile activity. FS activates NO(i) production from both eNOS and nNOS, and each source of NO(i) exerts opposing effects on [Ca(2+)](i) transient amplitude. These findings are important for understanding the regulation of NO(i) signalling in the normal and mechanically failing heart.

Combined antiretroviral therapy causes cardiomyopathy and elevates plasma lactate in transgenic AIDS mice.

Highly active antiretroviral therapy (HAART) is implicated in cardiomyopathy (CM) and in elevated plasma lactate (LA) in AIDS through mechanisms of mitochondrial dysfunction. To determine mitochondrial events from HAART in vivo, 8-week-old hemizygous transgenic AIDS mice (NL4-3Delta gag/pol; TG) and wild-type FVB/n littermates were treated with the HAART combination of zidovudine, lamivudine, and indinavir or vehicle control for 10 days or 35 days. At termination of the experiments, mice underwent echocardiography, quantitation of abundance of molecular markers of CM (ventricular mRNA encoding atrial natriuretic factor [ANF] and sarcoplasmic calcium ATPase [SERCA2]), and determination of plasma LA. Myocardial histologic features were analyzed semiquantitatively and results were confirmed by transmission electron microscopy. After 35 days in the TG + HAART cohort, left ventricular mass increased 160% by echocardiography. Molecularly, ANF mRNA increased 250% and SERCA2 mRNA decreased 57%. Biochemically, LA was elevated (8.5 +/- 2.0 mM). Pathologically, granular cytoplasmic changes were found in cardiac myocytes, indicating enlarged, damaged mitochondria. Findings were confirmed ultrastructurally. No changes were found in other cohorts. After 10 days, only ANF was elevated, and only in the TG + HAART cohort. Results show that cumulative HAART caused mitochondrial CM with elevated LA in AIDS transgenic mice.

Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C epsilon and delta in neonatal rat ventricular myocytes.

Protein kinase C (PKC) epsilon and PKCdelta translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38(MAPK) cascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38(MAPK) activity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKCepsilon and PKCdelta were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38(MAPK) in NRVMs. Adv-caPKCepsilon infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKCepsilon levels in a time- and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKCepsilon induced a time- and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding beta-galactosidase (Adv-nebetagal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38(MAPK) was relatively unaffected. Adv-caPKCdelta infection (1 to 25 MOI, 4 to 48 hours) increased total PKCdelta levels in a similar fashion. Adv-caPKCdelta (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38(MAPK) 24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKCdelta, but not Adv-caPKCepsilon, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner.

Pyk2 expression and phosphorylation in neonatal and adult cardiomyocytes.

A. L. Bayer, A. G. Ferguson, P. A. Lucchesi and A. M. Samarel. PYK2 Expression and Phosphorylation in Neonatal and Adult Cardiomyocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 1017-1030. Proline-rich tyrosine kinase (PYK2) is a Ca(2+)-dependent, non-receptor protein tyrosine kinase involved in growth factor signaling. Although PYK2 is expressed in a variety of tissues, it has not yet been identified in cardiac muscle. Therefore, immunocytochemical and Western blotting techniques were used to examine PYK2 expression and phosphorylation in neonatal and adult rat ventricular cardiomyocytes (NRVM and ARVM, respectively). PYK2 concentration was much greater in neonatal, than in adult ventricular tissue and cardiomyocytes. In cultured cells, PYK2 expression was highly dependent on [Ca(2+)](i)transients and contractile activity. Non-contracting, low-density NRVM in serum-free culture expressed very low levels of PYK2, while high-density, spontaneously contracting NRVM showed a approximately 12-fold increase in PYK2 expression. Conversely, high-density NRVM treated with nifedipine (10 microM, 48 h) to block spontaneous [Ca(2+)](i)transients and contractile activity resulted in a 2.6-fold decrease in PYK2 levels. Similarly, overnight culture of quiescent ARVM markedly reduced PYK2 levels. Chronic treatment (48 h) of cultured NRVM with the hypertrophic agonist endothelin-1 (ET) (10-300 n M) did not significantly increase PYK2 levels, but strongly shifted the ratio of phosphorylated to total PYK2, indicating that PYK2 phosphorylation accompanies cardiomyocyte hypertrophy. Endothelin-1 also acutely activated PYK2 in both cultured NRVM, and in freshly isolated ARVM. These results suggest that PYK2 is involved in the generation of certain aspects of cardiomyocyte hypertrophy.

Role of protein kinase C-epsilon in hypertrophy of cultured neonatal rat ventricular myocytes.

Using adenovirus (Adv)-mediated overexpression of constitutively active (ca) and dominant-negative (dn) mutants, we examined whether protein kinase C (PKC)-epsilon, the major novel PKC isoenzyme expressed in the adult heart, was necessary and/or sufficient to induce specific aspects of the hypertrophic phenotype in low-density, neonatal rat ventricular myocytes (NRVM) in serum-free culture. Adv-caPKC-epsilon did not increase cell surface area or the total protein-to-DNA ratio. However, cell shape was markedly affected, as evidenced by a 67% increase in the cell length-to-width ratio and a 17% increase in the perimeter-to-area ratio. Adv-caPKC-epsilon also increased atrial natriuretic factor (ANF) and beta-myosin heavy chain (MHC) mRNA levels 2.5 +/- 0.3- and 2.1 +/- 0.2-fold, respectively, compared with NRVM infected with an empty, parent vector (P < 0.05 for both). Conversely, Adv-dnPKC-epsilon did not block endothelin-induced increases in cell surface area, the total protein-to-DNA ratio, or upregulation of beta-MHC and ANF gene expression. However, the dominant-negative inhibitor markedly suppressed endothelin-induced extracellular signal-regulated kinase (ERK) 1/2 activation. Taken together, these results indicate that caPKC-epsilon overexpression alters cell geometry, producing cellular elongation and remodeling without a significant, overall increase in cell surface area or total protein accumulation. Furthermore, PKC-epsilon activation and downstream signaling via the ERK cascade may not be necessary for cell growth, protein accumulation, and gene expression changes induced by endothelin.

Brief rapid pacing depresses contractile function via Ca(2+)/PKC-dependent signaling in cat ventricular myocytes.

The purpose of this study is to determine the effects of brief rapid pacing (RP; approximately 200-240 beats/min for approximately 5 min) on contractile function in ventricular myocytes. RP was followed by a sustained inhibition of peak systolic cell shortening (-44 +/- 4%) that was not due to changes in diastolic cell length, membrane voltage, or L-type Ca(2+) current (I(Ca,L)). During RP, baseline and peak intracellular Ca(2+) concentration ([Ca(2+)](i)) increased markedly. After RP, Ca(2+) transients were similar to control. The effects of RP on cell shortening were not prevented by 1 microM calpain inhibitor I, 25 microM L-N(5)-(1-iminoethyl)-orthinthine, or 100 microM N(G)-monomethyl-L-arginine. However, RP-induced inhibition of cell shortening was prevented by lowering extracellular [Ca(2+)] (0.5 mM) during RP or exposure to chelerythrine (2-4 microM), a protein kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective inhibitor of PKC-beta. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (-46 +/- 7%). Western blots indicated that cat myocytes express PKC-alpha, -delta, and -epsilon as well as PKC-beta. These findings suggest that brief RP of ventricular myocytes depresses contractility at the myofilament level via Ca(2+)/PKC-dependent signaling. These findings may provide insight into the mechanisms of contractile dysfunction that follow paroxysmal tachyarrhythmias.

Focal adhesion kinase is involved in angiotensin II-mediated protein synthesis in cultured vascular smooth muscle cells.

The rate of vascular smooth muscle cell protein synthesis and cellular hypertrophy in response to angiotensin II (Ang II) is dependent on activation of protein tyrosine kinases (PTKs) and both the extracellular signal-regulated kinase (ERK) 1/2 and p70(S6K) pathways. One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions. We used an actin depolymerizing agent, cytochalasin D (Cyt-D), and a replication-defective adenovirus encoding FAK-related nonkinase (FRNK), an inhibitor of FAK-dependent signaling, as tools to assess whether FAK was upstream of the ERK1/2 and/or the p70(S6K) pathways. Cyt-D reduced basal FAK phosphorylation and blocked Ang II-dependent FAK phosphorylation in a dose-dependent manner. Confocal microscopy indicated that Cyt-D induced actin filament disruption and FAK delocalization from focal adhesions. Cyt-D also reduced Ang II-induced ERK1/2 activation, but p70(S6K) activation was relatively unaffected. Cyt-D reduced basal protein synthetic rate and substantially reduced the Ang II-induced increase in protein synthesis. Similarly, FRNK overexpression blocked Ang II-induced FAK phosphorylation and ERK1/2 activation, but not p70(S6K) phosphorylation, and markedly inhibited protein synthesis. This is the first report to demonstrate that FAK is a critical component of the signal transduction pathways that mediate Ang II-induced ERK1/2 activation, c-fos induction, and enhanced protein synthesis in vascular smooth muscle cells.

Laminin binding to beta1-integrins selectively alters beta1- and beta2-adrenoceptor signalling in cat atrial myocytes.

1. Perforated patch recordings were used to determine how plating atrial cells on laminin alters beta-adrenergic receptor (beta-AR) regulation of L-type Ca2+ current (ICa,L). 2. Isoproterenol (isoprenaline; ISO; 0.01 microM), a non-selective beta-AR agonist, elicited a greater stimulation of ICa,L in cells plated on laminin (+79 +/- 16 %; n = 17) than on glass (+33 +/- 5 %; n = 23). Also, desensitization to ISO was greater in cells on laminin (-16 +/- 2 %) than on glass (-3 +/- 1 %). Atenolol (0.1 microM), a selective beta1-AR antagonist, inhibited the effects of ISO in cells on glass but not laminin. Conversely, 0.1 microM ICI 118,551, a selective beta2-AR antagonist, inhibited the effects of ISO in cells on laminin but not glass. With beta2-ARs blocked, ISO-induced stimulation of ICa,L was greater in cells on glass than laminin. 3. Zinterol (0.01-0.1 microM), a selective beta2-AR agonist, elicited a greater stimulation of ICa,L in cells on laminin than on glass. The effects of zinterol were blocked by ICI 118,551. 4. ISO-induced stimulation of ICa,L was greater in cells plated on an alphabeta1-integrin antibody than on glass. Also, addition of 20 microM cytochalasin D to cells on laminin prevented the enhanced effects of ISO typically elicited in cells on laminin alone. 5. We conclude that laminin binding to alphabeta1-integrins, in conjunction with the actin cytoskeleton, reduces beta1-AR and enhances beta2-AR signalling which regulates ICa,L. This novel mechanism may contribute to remodelling of beta-AR signalling in the failing heart.

Laminin acts via beta 1 integrin signalling to alter cholinergic regulation of L-type Ca(2+) current in cat atrial myocytes.

A perforated patch recording method was used to determine how plating cells on laminin (20 microg ml(-1); >2 h) alters cholinergic regulation of L-type Ca(2+) current (I(Ca,L)) in atrial myocytes. Acetylcholine (ACh; 1 microm)-induced inhibition of basal I(Ca,L) was not different between cells on glass and laminin. However, stimulation of I(Ca,L) elicited by ACh withdrawal was significantly smaller in cells on laminin (10 +/- 2 %) than on glass (48 +/- 5 %) (P < 0.001). Stimulation of I(Ca,L) induced by either spermine-NO (200 microm), milrinone (10 microm), IBMX (100 microm) or forskolin (1 microm) was significantly smaller in cells plated on laminin than on glass. However, stimulation of I(Ca,L) by 100 microm 8-CPT-cAMP or intracellular dialysis with 50 microM cAMP was not different between cells plated on laminin or glass. Basal, forskolin- and IBMX-stimulated cAMP content was significantly smaller in cells plated on laminin than on glass. Stimulation of I(Ca,L) by ACh withdrawal was significantly smaller in cells plated on an alpha beta 1-integrin antibody (10 +/- 4 %) than on glass (3 +/- 6 %; P < 0.001). In cells on laminin, prior exposure to 100 microg ml-1 YIGSR, a laminin receptor-binding peptide, restored ACh-induced stimulation of I(Ca,L) (58 +/- 14 %)laminin alone (7 +/- 2 %; P < 0. 05). Addition of 20 microm cytochalasin D or 1 microM latrunculin A, agents that prevent actin polymerization, to cells on laminin restored ACh-induced stimulation of I(Ca,L). We conclude that laminin binding to beta 1 integrins acts in association with the actin-based cytoskeleton to attenuate adenylate cyclase activity. As a result, laminin inhibits NO-mediated stimulation of I(Ca,L) elicited by ACh withdrawal. Laminin-integrin signalling may be relevant to changes in autonomic regulation that occur during cardiac development and/or disease.

Isoenzyme-specific protein kinase C and c-Jun N-terminal kinase activation by electrically stimulated contraction of neonatal rat ventricular myocytes.

Previous studies from our laboratory and others indicate that contraction-induced mechanical loading of cultured neonatal rat ventricular myocytes produces many of the phenotypic changes associated with cardiomyocyte hypertrophy in vivo, and that these changes occur via the activation of serine-threonine protein kinases. These may include the extracellular regulated protein kinases (ERK1 and ERK2), the c-Jun N-terminal kinases (JNK1, JNK2, and JNK3), and one or more isoenzymes of protein kinase C. In this study, we assessed whether one or more of these kinases are activated by stimulated contraction, and whether activation was isoenzyme-specific. Low-density, quiescent cultures of neonatal rat ventricular myocytes were maintained in serum-free medium, or electrically stimulated to contract (3 Hz) for up to 48 h. ERK and JNK activation was assessed by Western blotting with polyclonal antibodies specific for the phosphorylated forms of both kinases. PKC activation was analysed by subcellular fractionation, detergent extraction, and Western blotting using isoenzyme-specific monoclonal antibodies. Stimulated contractile activity produced myocyte hypertrophy, as indicated by increased cell size, a 15+/-5% increase in total protein/DNA ratio, and induction of ANF and beta MHC gene transcription. Electrical pacing did not cause ERK1/2 or JNK1 activation, but increased JNK2 and JNK3 phosphorylation by;two-fold. Subcellular fractionation revealed a time-dependent increase in PKC delta, and to a much lesser extent PKC xi, in a Triton X-100-soluble membrane fraction within 5 min of the onset of stimulated contraction. PKC alpha was not activated by electrical pacing. These results indicate that contraction-induced mechanical loading acutely activates some but not all of the specific isoenzymes of JNKs and PKCs in cardiomyocytes.

Endothelin-induced cardiac myocyte hypertrophy: role for focal adhesion kinase.

Endothelin-1 (ET) produces neonatal rat ventricular myocyte (NRVM) hypertrophy and activates focal adhesion kinase (FAK) in other cell types. In the present study, we examined whether ET activated FAK in NRVM and whether FAK was necessary and/or sufficient for ET-induced NRVM hypertrophy. Chronic ET-1 stimulation (100 nM, 48 h) increased protein-to-DNA and myosin heavy chain (MHC)-to-DNA ratios and stimulated the assembly of newly synthesized MHC into sarcomeres. ET-1 also induced the assembly of focal adhesions and costameres, as evidenced by increased phosphotyrosine, FAK, and paxillin immunostaining. Acutely, ET treatment rapidly increased tyrosine phosphorylation of FAK and paxillin. FAK was also activated by phorbol 12-myristate 13-acetate (2 microM, 5 min). Pretreatment with chelerythrine (5 microM) or rottlerin (10 microM) completely blocked ET-induced FAK phosphorylation, indicating that protein kinase C activation was upstream of ET-induced FAK activation. In contrast, ET-induced FAK activation was not affected by blocking calcium influx via L-type voltage-gated calcium channels. Adenoviruses (Adv) containing FAK and FAK-related nonkinase (FRNK) were used to specifically define the role of FAK in ET-induced hypertrophy. ET stimulation failed to increase total protein-to-DNA or MHC-to-DNA ratios or to stimulate sarcomeric assembly in myocytes infected with Adv-FRNK. However, Adv-FAK alone did not increase total protein-to-DNA or MHC-to-DNA ratios and failed to increase the number or size of myofibrils as evidenced by double immunofluorescence labeling for MHC and FAK. Thus, although FAK is necessary for ET-induced NRVM hypertrophy, other ET-generated signals are also required to elicit the hypertrophic phenotype.

Cardiac dysfunction occurs in the HIV-1 transgenic mouse treated with zidovudine.

Cardiomyopathy in AIDS is an increasingly important clinical problem. Mechanisms of AIDS cardiomyopathy were explored using AIDS transgenic mice that express replication-incompetent HIV-1 (NL4-3delta gag/pol). Transgenic and FVB/n mice (n = 3 to 6 per cohort) received water ad libitum with and without zidovudine (3'-azido-2',3'-deoxythymidine; AZT; 0.7 mg/ml) for 21 or 35 days. After 21 days, echocardiographic studies were performed and abundance of mRNA for cardiac sarcoplasmic reticulum calcium ATPase (SERCA2), sodium calcium exchanger (NCX1), and atrial natriuretic factor were determined individually using Northern analysis of extracts of left ventricles. After 35 days, contractile function and relaxation were analyzed in isolated work-performing hearts. Histopathological and ultrastructural (transmission electron microscopy) changes were identified. After 21 days, molecular indicators of cardiac dysfunction were found. Depressed SERCA2 and increased atrial natriuretic factor mRNA abundance occurred in left ventricles from AZT-treated transgenic mice. NCX1 abundance was unchanged. Eccentric left ventricle hypertrophy was determined echocardiographically. After 35 days, cardiac dysfunction was worst in AZT-treated and AZT-untreated transgenic mice. Decreases in the first derivative of the maximal change in left ventricle systolic pressure with respect to time (+dP/dt) occurred in transgenic mice with and without AZT. Increased half-time of relaxation and ventricular relaxation (-dP/dt) occurred in AZT-treated and -untreated transgenic mice. Increased time to peak pressure was found only in AZT-treated transgenic mice. In AZT-treated FVB/n mice, -dP/dt was decreased. Ultrastructurally, mitochondrial destruction was most pronounced in AZT-treated transgenic mice, but also was found in AZT-treated FVB/n mice. Transgenic mice that express HIV-1 demonstrate cardiac dysfunction. AZT treatment of FVB/n mice causes mitochondrial ultrastructural alterations that are similar to those in other species. In transgenic mice, AZT treatment worsens molecular and ultrastructural features of cardiomyopathy. HIV-1 constructs and AZT each contribute to cardiac dysfunction in this murine model of AIDS cardiomyopathy.

Upregulation of Na(+)/Ca(2+) exchanger expression and function in an arrhythmogenic rabbit model of heart failure.

Three-dimensional cardiac mapping in rabbits with nonischemic cardiomyopathy has shown that ventricular arrhythmias initiate by a nonreentrant mechanism that may be due to triggered activity from delayed afterdepolarizations. Delayed afterdepolarizations are thought to be due to spontaneous release of Ca(2+) from the sarcoplasmic reticulum (SR) and consequent activation of an inward Na(+)/Ca(2+) exchange (NaCaX) current. The goal of this study was to determine whether there is enhanced NaCaX gene expression and functional activity that may contribute to nonreentrant activation. Heart failure (HF) was induced in rabbits by combined aortic insufficiency and aortic constriction. HF rabbits had left ventricular enlargement (left ventricular end-diastolic dimension increased from 1.43+/-0.03 to 1.97+/-0.05 cm) and severely depressed function (fractional shortening reduced from 37% to 26%, P<0.02). Heart-to-body weight was increased by 79% in HF. Western blots showed a 93% increase in NaCaX protein in HF (P<0.04). NaCaX mRNA (7-kb transcript) was increased by 104% relative to the 18S rRNA in HF. A 14-kb NaCaX transcript was also seen in the HF rabbits, raising total NaCaX mRNA to 2.7-fold compared with controls. The amplitude of caffeine-induced contractures, used to assess SR Ca(2+) load, was not significantly different in HF. Relaxation and [Ca(2+)](i) decline during caffeine-induced contractures is attributable to Ca(2+) transport by NaCaX and was 61% and 45% faster in HF (P<0.05), respectively. NaCaX current measured under controlled voltage clamp conditions was also 2-fold higher in HF cells. SR Ca(2+)-ATPase mRNA and protein levels and Ca(2+) current density were not significantly altered in HF. Twitch amplitudes from HF myocytes were 26% smaller compared with control (P<0.02), but twitch relaxation and [Ca(2+)](i) decline (due largely to SR Ca(2+)-ATPase) were not altered. Thus myocytes and myocardium from HF rabbits exhibit enhanced NaCaX expression and function. The enhanced NaCaX activity may contribute to depressed contractions, increased transient inward current (for a given SR Ca(2+) release), delayed afterdepolarizations, and nonreentrant initiation of ventricular tachycardia in this arrhythmogenic model of HF.