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.

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.

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.

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.

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.

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 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.

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.

Sarcomeric myosin heavy chain is degraded by the proteasome.

Cardiac myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The balance between these opposing metabolic processes ultimately determines the number of functional contractile units within each cardiac muscle cell. Although alterations in myofibrillar protein degradation have been shown to contribute to cardiac growth and remodeling, the intracellular proteolytic systems responsible for degrading myofibrillar proteins to their constitutive amino acids are currently unknown. Lactacystin, a recently developed, highly specific proteasome inhibitor, was used in this study to examine the role of the proteasome in myosin heavy chain (MHC) degradation in cultured neonatal rat ventricular myocytes. Cells were treated with growth medium alone or with lactacystin (1-50 microM) for up to 48 h. Lactacystin significantly increased the total protein/DNA ratio and markedly prolonged MHC half-life. Other proteasome inhibitors, namely carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (10 microM) and N-acetyl-L-leucyl-L-leucyl-norleucinal (100 microM), were also effective in suppressing MHC degradation. Lactacystin and other proteasome inhibitors also suppressed the markedly accelerated MHC degradation associated with Ca2+ channel blockade but did not prevent the disassembly and loss of myofibrils that accompanied contractile arrest. Thus, sarcomere disassembly precedes the degradation of MHC, which is at least in part mediated by the proteasome.

Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes.

Left ventricular hypertrophy (approximately 40%) was induced in rats by banding of the abdominal aorta. After 16 wk, ventricular homogenates were prepared for biochemical measurements and ventricular myocytes were isolated for functional studies. In myocytes, the effects of banding on intracellular Ca handling, contraction, and excitation-contraction (E-C) coupling were determined using indo 1 fluorescence and whole cell voltage clamp. After steady-state field or voltage-clamp stimulation to load the sarcoplasmic reticulum (SR), SR Ca content assessed by caffeine-induced Ca transients was the same in sham and banded groups. Despite this, cell shortening amplitudes were significantly depressed in the banded group, suggesting altered contractile properties. In banded rats, the SR Ca-adenosinetriphosphatase (Ca-ATPase) mRNA level was reduced, as was homogenate thapsigargin-sensitive SR Ca-ATPase, but cytosolic free Ca concentration ([Ca]i) decline attributed to SR Ca-ATPase activity in intact cells was not slowed. Banding also reduced Na/Ca exchange mRNA level but did not affect either Na-dependent sarcolemmal 45Ca transport in homogenate or the rate of [Ca]i decline in intact cells attributed to Na/Ca exchange (during caffeine-induced contractures). Banding also did not change the rate of [Ca]i decline mediated by the combined function of the mitochondrial Ca uptake and sarcolemmal Ca-ATPase in intact cells. Ca current (ICa) density and voltage dependence were the same in sham and banded groups. Ryanodine receptor mRNA, protein content, and ryanodine affinity were also unchanged in the banded group. At 1 mM extracellular Ca concentration ([Ca]o), banding did not affect E-C coupling efficacy in intact cells under voltage clamp (i.e., same contraction for given ICa and SR Ca load). However, when [Ca]o was reduced to 0.5 mM, the efficacy of E-C coupling was greatly depressed in the banded group (even though ICa and SR Ca content were matched). In summary, unloaded myocyte contraction was depressed in these hypertrophic hearts, but Ca transport was little altered, at 1 mM [Ca]o. However, reduction of [Ca]o to 0.5 mM appears to unmask a depressed fractional SR Ca release in response to a given ICa trigger and SR Ca load.

Cyclic stretch down-regulates calcium transporter gene expression in neonatal rat ventricular myocytes.

Abnormal intracellular Ca2+ handling in hypertrophied and failing hearts is partly due to changes in Ca2+ transporter gene expression, but the mechanisms responsible for these alterations remain largely unknown. We previously showed that intrinsic mechanical load (i.e. spontaneous contractile activity) induced myocyte hypertrophy, and down-regulated SR Ca2+ ATPase (SERCA2) gene expression in cultured neonatal rat ventricular myocytes (NRVM). In the present study, we examined whether extrinsic mechanical load (i.e. cyclic stretch) also induced NRVM hypertrophy, and led to down-regulation of SERCA2 and other Ca2+ transporter genes which have been associated with cardiac hypertrophy and failure in vivo. NRVM were maintained in serum-free culture medium under control conditions, or subjected to cyclic mechanical deformation (1.0 Hz, 20% maximal strain, 48 h). Under these conditions, cyclic stretch induced NRVM hypertrophy, as evidenced by significant increases in total protein/DNA ratio, myosin heavy chain (MHC) content, and atrial natriuretic factor (ANF) secretion. Cyclic stretch also induced the MHC isoenzyme "switch" which is characteristic of hemodynamic overload of the rat heart in vivo. Cyclic stretch significantly down-regulated SERCA2 and ryanodine receptor (RyR) mRNA and protein levels, while simultaneously increasing ANF mRNA. In contrast, Na+-Ca2+ exchanger and phospholamban mRNA levels were unaffected. Load-dependent SERCA2 and RyR down-regulation was independent of Ca2+ influx via voltage-gated, L-type Ca2+ channels, as cyclic stretch down-regulated SERCA2 and RyR mRNA levels in both control and verapamil-treated NRVM. These results indicate that extrinsic mechanical load (in the absence of other exogenous stimuli) induces NRVM hypertrophy and causes down-regulation of Ca2+ transporter gene expression. This in vitro model system should prove useful to dissect the intracellular signaling pathways responsible for transducing this phenotype during cardiac hypertrophy and heart failure in vivo.

Downregulation of sarcoplasmic reticulum Ca(2+)-ATPase during progression of left ventricular hypertrophy.

To determine whether reduced sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) (SERCA2) activity contributes to delayed myocardial relaxation during chronic left ventricular hypertrophy (LVH) progression, LVH was produced in rats by abdominal aortic coarctation. Systolic and diastolic functions were assessed in vivo 8 and 16 wk after surgery, and compositional alterations in LV myocardium [SERCA2 concentration, myosin heavy chain (MHC) isoenzymes, and tissue collagen] were correlated with the development of prolonged isovolumic relaxation and impaired cardiac performance over time. Myocardial relaxation was prolonged in 8-wk banded rats, despite normal isovolumic systolic function and LV end-diastolic pressure (LVEDP). No significant alterations in SERCA2 protein, beta-MHC, or fibrillar collagen levels were observed at this early time point. In contrast, LV SERCA2, beta-MHC, and fibrillar collagen concentrations were all significantly altered in 16-wk banded rats. These late compositional changes were associated with reduced cardiac performance, as manifested by a significant elevation in LVEDP (14 +/- 2 mmHg). The 34% decrease in SERCA2 protein was associated with reduced SR Ca2+ uptake and an even greater reduction (76%) in SERCA2 mRNA. SERCA2 mRNA levels were also significantly reduced to 43 +/- 10% of sham-operated rats 8 wk after banding, despite unchanged SERCA2 protein levels and normal SR Ca2+ uptake. These results argue against a significant contribution of SERCA2 downregulation to the subtle alterations in myocardial relaxation observed in compensated LVH. However, the early reduction in SERCA2 mRNA levels may serve as a molecular marker for impaired cardiac performance during the transition from compensated LVH to heart failure.

Phorbol 12-myristate 13-acetate alters SR Ca(2+)-ATPase gene expression in cultured neonatal rat heart cells.

Primary cultures of neonatal rat ventricular myocytes were used to examine how the cardiac myocyte cytoplasmic Ca2+ ([Ca2+]i) transient and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) gene expression change in response to treatment with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). Exposure of neonatal myocytes to PMA (200 nM, 48-72 h) produced myocyte growth and a 70% prolongation of the half-time for [Ca2+]i decline induced by potassium depolarization in the absence of extracellular Na+ (in which the sarcoplasmic reticulum Ca2+ pump is the main mechanism responsible for [Ca2+]i decline). The reduced rate of [Ca2+]i transient decline corresponded to a 53% reduction in SERCA2 protein levels and a 43% reduction in SERCA2 mRNA levels as compared with control myocytes. Exposure to PMA for as little as 30 min or for as long as 48 h produced a similar degree of SERCA2 mRNA downregulation over time. PMA-induced downregulation of SERCA2 mRNA levels was blocked by either 10 nM staurosporine or 4 microM chelerythrine, whereas treatment with either agent alone increased SERCA2 mRNA levels as compared with control cells. Actinomycin D mRNA stability assays revealed that PMA treatment appeared to markedly destabilize the relatively long-lived SERCA2 mRNA transcript. Taken together, these results indicate that downregulation of SERCA2 gene by PMA in cultured neonatal myocytes occurs at least in part by alterations in mRNA stability and results in functional alterations in [Ca2+]i decline that are similar to that observed in the hypertrophied and failing adult myocardium.

Alterations in skeletal muscle gene expression in the rat with chronic congestive heart failure.

Congestive heart failure is often associated with skeletal muscle abnormalities that contribute to early fatigue and acidosis. Up to the present time, however, the mechanisms responsible for these changes are unclear. Myocardial infarctions were produced by coronary ligation in adult Sprague-Dawley rats. At 20 weeks, 10 control rats, and 15 animals with heart failure [defined by elevated LVEDP (26.1 +/- 3.1 v 2.5 +/- 0.5 mmHg) and RV hypertrophy (300 +/- 21 g v 158 +/- 9 mg)] underwent in vivo measurements of total body, and soleus total protein and myosin heavy chain (MHC) synthesis by [3H]leucine constant infusion. Soleus muscle was also analysed for protein content, and MHC isoenzyme content by SDS-PAGE. Northern blotting also was used to determine levels of the mRNA's encoding type I, IIa, IIb, and IIx MHC, alpha-skeletal actin, COX III, SDH and GAPDH. Soleus muscles in heart failure rats were smaller than controls (112 +/- 6 v 126 +/- 5 mg) and the degree of atrophy was significant when corrected for body mass (0.38 +/- 0.02 v 0.46 +/- 0.02 mg/g. P = 0.007). Although there was no significant difference in plasma leucine flux (an index of whole-body protein synthesis), soleus muscle total and MHC synthesis was reduced in heart failure animals. Whereas the Type I MHC isoenzyme (beta MHC) was the only MHC detected in the soleus of control animals, type II MHC isoenzyme comprised 11.8 +/- 3.1% of the MHC in the heart failure group. Furthermore, steady-state mRNA levels encoding beta MHC were significantly depressed in the heart failure rats, where those encoding Types IIb and IIx MHC were increased. Steady-state mRNA levels of alpha-skeletal actin, cytochrome C oxidase (COX III) and succinate dehydrogenase (SDH) were also significantly depressed. This animal model of chronic heart failure is associated with quantitative and qualitative alterations in skeletal muscle gene expression that are similar to those reported in skeletal muscle of patients with chronic heart failure. The altered phenotype and impaired metabolic capacity may contribute to exercise intolerance in CHF.

Prolyl hydroxylation regulates intracellular procollagen degradation in cultured rat cardiac fibroblasts.

To determine the regulatory role of prolyl hydroxylation in intracellular cardiac procollagen turnover, we examined the effects of prolyl 4-hydroxylase inhibitors (alpha, alpha-dipyridil, 3,4-dihydroxybenzoic acid ethyl ester, pyridine 2,4-dicarboxylic acid ethyl ester) and ascorbic acid on procollagen metabolism by cultured, neonatal rat cardiac fibroblasts. Ascorbate-deficient fibroblasts showed decreased rates of prolyl hydroxylation and total collagen accumulation without a significant reduction in alpha 1(I) and alpha 1(III) mRNA levels. The fraction of newly synthesized procollagens degraded intracellularly was also substantially increased in ascorbate-deficient cells (50 +/- 7 v 30 +/- 3% in ascorbate-deficient v control fibroblasts; P < 0.05). These findings were associated with increased intracellular accumulation of Type I procollagen, enhanced secretion of "underhydroxylated" pro alpha 1 (I) polypeptide into the cell culture medium, and decreased extracellular Type I collagen deposition. Similar results were obtained by treating cells with alpha, alpha-dipyridil (300 microns), and 3,4-dihydroxybenzoic acid ethyl ester (400 microM) in the presence of ascorbate. A major portion of the enhanced degradation of newly synthesized procollagens occurred within acidic intracellular compartments as indicated by the inhibition of procollagen degradation by chloroquine (25 microM). Inhibition of procollagen secretion by colchicine (0.5 micrograms/ml) enhanced the diversion to, and subsequent intracellular degradation of underhydroxylated procollagens in cardiac fibroblast lysosomes. We conclude that inactivation of prolyl 4-hydroxylase increases intracellular accumulation and intralysosomal degradation of newly synthesized cardiac procollagen polypeptides. These observations suggest that procollagen prolyl hydroxylation may be important in the regulation of collagen accumulation by cardiac interstitial cells during fibrotic processes in vivo

Regulation of rat ventricular myosin heavy chain expression by serum and contractile activity.

To quantitatively analyze the effects of serum stimulation and contractile activity and their interaction on cellular growth and cardiac myosin heavy chain (MHC) gene expression, spontaneously contracting neonatal rat ventricular myocytes in primary culture were maintained in serum-free growth medium or growth medium supplemented with fetal bovine serum. Contractile activity in paired cultures was inhibited by addition of the calcium channel blocker verapamil (10 microM) to the culture medium. Both serum stimulation and contractile activity produced myocyte hypertrophy as assessed by increases in total protein, total RNA, protein-to-DNA ratios, and total MHC protein content. MHC isoenzyme analysis indicated that both MHC-alpha and MHC-beta proteins accumulated in response to serum stimulation and/or contractile activity. The increases in MHC-beta protein resulting from serum stimulation and contractile activity occurred in parallel with increases in MHC-beta mRNA. In contrast, MHC-alpha mRNA levels were relatively unaffected by serum stimulation but appeared to decrease in response to contractile activity. The protein kinase inhibitor staurosporine (5 nM) reduced MHC-beta expression in serum-free, contracting cultures and also prevented the serum-induced increase in MHC-beta mRNA observed in both contracting and arrested myocytes. Staurosporine also increased MHC-alpha mRNA levels in serum-free, contracting, and verapamil-arrested myocytes. These data suggest that both humoral and mechanical factors regulate MHC isoenzyme expression and cellular growth in neonatal ventricular myocytes.

Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells.

We developed protocols with intact cultured neonatal rat myocytes to directly evaluate the function of the sarcoplasmic reticulum (SR) Ca-ATPase (or SERCA2), Na-Ca exchange (Na-CaX), and slow Ca transport systems (mitochondria and sarcolemmal Ca-ATPase). Spontaneously beating control cells were compared with cells cultured for 2 days in the presence of verapamil (verapamil-arrested cells, VA). Intracellular calcium (Cai) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Nao), and (3) twitches induced by brief depolarizations with high [K]o solution (K-twitches). We also measured mRNA levels for the SR Ca-ATPase and Na-CaX in the same experimental preparations. The t1/2 for [Ca]i decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (approximately 20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t1/2 of CafC when Na-CaX was the main mechanism responsible for [Ca]i decline (t1/2 approximately 1.5 seconds), indicating unaltered Na-CaX. Conversely, we found nearly a twofold increase in the rate of [Ca]i decline during K-twitches (control t1/2, 0.84 +/- 0.05 seconds; VA t1/2, 0.48 +/- 0.06 second; P < .001), indicating an increase in SR Ca-pumping activity in VA cells. This was also reflected by a 56% increase in the peak [Ca]i reached during CafC used to assess maximal SR Ca content (427 +/- 49 nmol/L in control versus 665 +/- 75 nmol/L in VA cells).(ABSTRACT TRUNCATED AT 250 WORDS)