Intracellular signaling pathways required for rat vascular smooth muscle cell migration. Interactions between basic fibroblast growth factor and platelet-derived growth factor.

Intracellular signaling pathways activated by both PDGF and basic fibroblast growth factor (bFGF) have been implicated in the migration of vascular smooth muscle cells (VSMC), a key step in the pathogenesis of many vascular diseases. We demonstrate here that, while bFGF is a weak chemoattractant for VSMCs, it is required for the PDGF-directed migration of VSMCs and the activation of calcium/calmodulin-dependent protein kinase II (CamKinase II), an intracellular event that we have previously shown to be important in the regulation of VSMC migration. Neutralizing antibodies to bFGF caused a dramatic reduction in the size of the intracellular calcium transient normally seen after PDGF stimulation and inhibited both PDGF-directed VSMC migration and CamKinase II activation. Partially restoring the calcium transient with ionomycin restored migration and CamKinase II activation as did the forced expression of a mutant CamKinase II that had been "locked" in the active state by site-directed mutagenesis. These results suggest that bFGF links PDGF receptor stimulation to changes in intracellular calcium and CamKinase II activation, reinforcing the central role played by CamKinase II in regulating VSMC migration.

Excitation-contraction coupling in heart: new insights from Ca2+ sparks.

Ca2+ sparks, the elementary units of sarcoplasmic reticulum (SR) Ca2+ release in cardiac, smooth and skeletal muscle are localized (2-4 microns ) increases in intracellular Ca2+ concentration, [Ca2+]i, that last briefly (30-100 ms). These Ca2+ sparks arise from the openings of a single SR Ca2+ release channel (ryanodine receptor, RyR) or a few RyRs acting in concert. In heart muscle, Ca2+ sparks can occur spontaneously in quiescent cells at a low rate (100 s-1 per cell). Identical Ca2+ sparks are also triggered by depolarization because the voltage-gated sarcolemmal L-type Ca2+ channels (dihydropyridine receptors, DHPRs) locally increase [Ca2+]i and thereby activate the RyRs by Ca(2+)-induced Ca2+ release (CICR). The exquisite responsiveness of this process, reflected by the ability of even a single DHPR to activate a Ca2+ spark, is perhaps due to the large local increase in [Ca2+]i in the vicinity of the RyR that is a consequence of the close apposition of the DHPRs and the RyRs. In this review we examine our current understanding of cardiac excitation-contraction (EC) coupling in light of recent studies on the elementary Ca2+ release events or Ca2+ sparks. In addition, we further characterized Ca2+ spark properties in rat and mouse heart cells. Specifically we have determined that: (i) Ca2+ sparks occur at the junctions between the transverse-tubules and the SR in both species; (ii) Ca2+ sparks are asymmetric, being 18% longer in the longitudinal direction than in the transverse direction; and (iii) Ca2+ sparks individually do not produce measurable sarcomere shortening (< 1%). These results are discussed with respect to local activation of the RyRs, the stability of CICR, Ca2+ diffusion, and the theory of EC coupling.

Effect of U-50,488H on the contractile response of cardiomyopathic hamster ventricular myocytes.

We examined the effects of a selective kappa opioid receptor agonist (U-50,488H) on the contractile properties of single ventricular myocytes from 127 day old control (F1B) and cardiomyopathic (BIO 14.6) hamsters. Myocytes in bicarbonate buffered solution with 1.5 mM [Ca2+] were electrically stimulated with field electrodes in the bath. Length changes were monitored via myocyte edge tracking. Twitch amplitude and the velocity of cell shortening were less in the cardiomyopathic hamster myocytes than in age-matched hamsters (P less than or equal to 0.05). There was a concentration-dependent effect of U-50,488H (0.1-20 microM) to decrease twitch amplitude and shortening velocity in both control and cardiomyopathic myocytes (P less than or equal to 0.001). In cells loaded with the Ca2+ indicator indo-1 the negative inotropic action of U-50,488H was associated with a decreased indo-1 fluorescence transient amplitude. There was no difference in the negative inotropic effect of U-50,488H on control and cardiomyopathic cells. Thus, the CM hamster does not demonstrate a different contractile response to U-50,488H.

Paracrine effects of hypoxic fibroblast-derived factors on the MPT-ROS threshold and viability of adult rat cardiac myocytes.

Cardiac fibroblasts contribute to multiple aspects of myocardial function and pathophysiology. The pathogenetic relevance of cytokine production by these cells under hypoxia, however, remains unexplored. With the use of an in vitro cell culture model, this study evaluated cytokine production by hypoxic cardiac fibroblasts and examined two distinct effects of hypoxic fibroblast-conditioned medium (HFCM) on cardiac myocytes and fibroblasts. Hypoxia caused a marked increase in the production of tumor necrosis factor (TNF)-alpha by cardiac fibroblasts. HFCM significantly enhanced the susceptibility of cardiac myocytes to reactive oxygen species (ROS)-induced mitochondrial permeability transition (MPT), determined by high-precision confocal line-scan imaging following controlled, photoexcitation-induced ROS production within individual mitochondria. Furthermore, exposure of cardiac myocytes to HFCM for 5 h led to loss of viability, as evidenced by change in morphology and annexin staining. HFCM also decreased DNA synthesis in cardiac fibroblasts. Normoxic fibroblast-conditioned medium spiked with TNF-alpha at 200 pg/ml, a concentration comparable to that in HFCM, promoted loss of myocyte viability and decreased DNA synthesis in cardiac fibroblasts. These effects of HFCM are similar to the reported effects of hypoxia per se on these cell types, showing that hypoxic fibroblast-derived factors may amplify the distinct effects of hypoxia on cardiac cells. Importantly, because both hypoxia and oxidant stress prevail in a setting of ischemia and reperfusion, the effects of soluble factors from hypoxic fibroblasts on the MPT-ROS threshold and viability of myocytes may represent a novel paracrine mechanism that could exacerbate ischemia-reperfusion injury to cardiomyocytes.

Novel technique to load indo-1 free acid into single adult cardiac myocytes to assess cytosolic Ca2+.

This report introduces a novel method to load indo-1 "free acid" selectively into the cytosol of cardiac myocytes, presumably by diffusion through momentarily permeable gap junction sites during mechanical dissociation after low-Ca2+ collagenase treatment. Calibration of indo-1 fluorescence in these cells has been accomplished after subtracting average autofluorescence (AF) from time-matched non-indo-loaded cells, taking into account apparent changes in cell AF due to indo-1. There is wide variation in the degree of uncertainty of individual intracellular Ca2+ concentration ([Ca2+]i) determinations among cells, related principally to differences in cellular indo-1 content, to nonlinear aspects of the [Ca2+]-to-fluorescence ratio relationship, and to the uncertainty in the AF subtraction. Consequently, a quantitative estimate of uncertainty also may be employed in formulating weighted estimates of cytosolic [Ca2+]i. The following [Ca2+]i values in rat ventricular cells (nM; in 1 mM bathing extracellular Ca2+ concentration, 25 degrees C) are given as weighted means +/- 95% confidence intervals (unweighted values in parentheses): 138 +/- 5 (136 +/- 6, n = 44) in quiescent cells, 435 +/- 74 (482 +/- 76, n = 43) at the [Ca2+]i-transient peak during 0.5 Hz steady-state stimulation, and 760 +/- 124 (1,027 +/- 250, n = 42) at the [Ca2+]i-transient peak, postrest. Moreover, these peak [Ca2+]i values fall near the steepest portion of the force-Ca2+ curve (from intact cardiac muscle), consistent with sensitive inotropic regulation and maximal contractile reserve.

L- and T-type calcium currents differ in finch and rat ventricular cardiomyocytes.

We compared L-type Ca current (ICaL) and T-type Ca current (ICaT) in finch and rat myocytes, using whole-cell patch clamp techniques. Cell capacitance averaged 50 +/- 4 pF in finch (n = 25) v 145 +/- 8 pF in rat (n = 38) cells, P < 0.001. In cells bathed in 1 mM Cao at 22 degrees C, peak ICaL amplitude, during a voltage clamp step (10 mM EGTA in pipette) from -45 mV to -5 mV, averaged 10.5 +/- 0.3 pA/pF in finch v 6.9 +/- 0.6 pA/pF, P < 0.001 in rat cells. ICaL inactivation kinetics were faster in finch (4.6 +/- 0.3 ms) than in rat (13.4 +/- 1.3 ms) cells. P < 0.001. ICaT was not detectable in rat cells (2 mM bathing [Ca]); but in finch cells, a large ICaT which averaged 6.8 +/- 1.4 pA/pF was activated at -30 mV and was relatively insensitive to nitrendipine (0.1 microM). The distinctive features of ICaL and ICaT in finch cells may have a role in the ability of the finch to achieve a very rapid heart rate. They may also facilitate excitation-Ca2+ release coupling in finch ventricular cells which are devoid of T tubules and have relatively few junctions between the sarcolemma and the sarcoplasmic reticulum.

Opposing effects of alpha 1-adrenergic receptor subtypes on Ca2+ and pH homeostasis in rat cardiac myocytes.

We examined the effect of alpha 1-adrenergic receptor (AR) subtypes on contraction, cytosolic Ca2+ concentration ([Ca2+]i), and cytosolic pH (pHi) of rat ventricular myocytes loaded with the Ca2+ indicator indo 1 or the pH indicator carboxyseminaphthorhodafluor-1. Nonselective alpha 1-AR stimulation was effected with phenylephrine plus nadolol. alpha 1-AR subtype stimulation was achieved with alpha 1-AR and chloroethylclonidine (CEC) or with alpha 1-AR and WB-4101. Cells were in bicarbonate buffer with 0.5 mM Ca2+ and were electrically stimulated at 0.5 Hz. Results show that 1) nonselective alpha 1-AR stimulation increased twitch and [Ca2+]i transient amplitudes, myofilament response to Ca2+, and pHi; 2) alpha 1-AR plus CEC increased twitch and [Ca2+]i transient amplitudes and also enhanced myofilament response to Ca2+ via cytosolic alkalinization; 3) alpha 1-AR plus WB-4101 decreased twitch and [Ca2+]i transient amplitudes and also pHi; and 4) cytosolic acidification due to alpha 1-AR plus WB-4101 was abolished by protein kinase C inhibition (staurosporine pretreatment) or downregulation (prolonged exposure to phorbol esters). In summary, the net effects of alpha 1-adrenergic stimulation on contraction, [Ca2+]i, and pHi are due to opposing WB-4101- and CEC-sensitive alpha 1-AR subtype signaling pathways.

Beta-receptor-adenylate cyclase coupling in hypoxic neonatal rat ventricular myocytes.

This study investigated the influence of hypoxia on alterations in the beta-adrenergic receptor-adenylate cyclase system. Cultured neonatal rat ventricular myocytes were subjected to normoxia (incubator PO2 135-145 mmHg) or hypoxia (incubator PO2 0-14 mmHg) and, in crude membrane preparations, beta-receptor binding properties were measured with [125I]iodocyanopindolol and adenylate cyclase activity by radioimmunoassay. Hypoxia of 30 min in duration caused no alteration in beta-receptor density (Bmax 75 +/- 11 vs. 71 +/- 12 fmol/mg protein) but increased adenylate cyclase activity under basal conditions and during stimulation with l-isoproterenol, 5'-guanylimidotriphosphate [Gpp(NH)p] 5 X 10(-5) M, NaF 10(-4) M, and forskolin 10(-4) M. For example, isoproterenol 10(-5) M + guanosine 5'-triphosphate (GTP) 5 X 10(-5) M gave 221 +/- 34 vs. 143 +/- 11 pmol.min-1.mg protein-1, P less than 0.05 hypoxia vs. normoxia. After 60 min of hypoxia, adenylate cyclase activity was no longer increased. Hypoxia of 120-150 min duration increased Bmax by 64% (73 +/- 8 to 120 +/- 11 fmol/mg protein, P less than 0.05 vs. normoxia) but decreased adenylate cyclase activity during stimulation with isoproterenol, NaF 10(-4) M, and forskolin 10(-4) M. For example, isoproterenol 10(-5) M + GTP 5 X 10(-5) M gave 107 +/- 12 vs. 148 +/- 11 pmol.min-1.mg protein-1, P less than 0.05 hypoxia vs. normoxia. Reoxygenation for 15 min following 120-150 min of hypoxia reversed the increased beta-receptor numbers and decreased adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Transgenic manipulation of beta-adrenergic receptor kinase modifies cardiac myocyte contraction to norepinephrine.

To determine the direct functional significance of the beta-adrenergic receptor (AR) kinase 1 (beta ARK1) on myocardial performance in the absence of tonic sympathoadrenal neural activation and mechanical loading, we measured the contractile responses to acute beta 1-AR stimulation in left ventricular myocytes isolated from nontransgenic control (NTG) and transgenic mice overexpressing either beta ARK1 (TG beta K12) or a beta ARK1 inhibitor (TGMini27). Contractile response to five concentrations (10(-8)-10(-7) M) of the beta 1-AR agonist norepinephrine (NE) plus prazosin (10(-6) M) was measured after a 60-s rest, i.e., rested-state contraction (RSC), and during steady-state contraction (SSC) stimulation at 0.5 Hz (23 degrees C). At baseline, resting cell length was significantly greater in TG beta K12 myocytes (P < 0.05); however, there were no significant differences in RSC or SSC among NTG, TG beta K12, or TG Mini27 mice. On the other hand, both the dose-response curve and kinetics for the NE-induced SSC response normalized to RSC (SSC/RSC) were significantly different among experimental groups (P < 0.001). Specifically, maximal SSC induced by NE in myocytes isolated from TG beta K12 was only 70% of the response observed in NTG cells and 50% of the response measured in TGMini27. These data suggest that 1) in the absence of circulating catecholamines or basal sympathetic tone, beta ARK1 actions in single myocytes are minimal, and 2) substantial functional beta ARK1 modulation of beta 1-AR signaling occurs in cardiac myocytes even during short-term beta 1-AR stimulation. These results are consistent with a role for agonist-induced phosphorylation and desensitization of cardiac beta 1-ARs by beta ARK1 in single myocytes and highlight the potential functional importance of beta ARK1 as a critical determinant of the cardiac beta 1-AR contractile response.

Beta 2-adrenergic receptor-stimulated increase in cAMP in rat heart cells is not coupled to changes in Ca2+ dynamics, contractility, or phospholamban phosphorylation.

Previous studies have shown that both beta 1- and beta 2-adrenergic receptors (AR) are present in rat ventricular myocytes, but stimulation of these receptor subtypes elicits qualitatively different cellular responses (Xiao, R.-P., and Lakatta, E. G. (1993) Circ. Res. 73, 286-300). In the present study, the biochemical mechanism underlying the distinct beta AR subtype actions have been investigated. Although both beta 1AR and beta 2AR stimulation increased total cellular cAMP in suspensions of rat ventricular myocytes to a similar extent, the maximum elevation of the membrane bound cAMP by beta 2AR stimulation was only half of that induced by beta 1AR stimulation, suggesting that stimulation the beta AR subtypes leads to different compartmentation of cAMP. The effects of beta 1AR stimulation on Ca2+ transient (indexed by the transient increase in indo-1 fluorescence ration after excitation) and contraction amplitude (measured via photodiode array) and their kinetics closely paralleled the increase in cAMP. In contrast, the increase in both membrane bound and total cAMP content after beta 2AR stimulation were completely dissociated from the effects of beta 2AR stimulation to increase the amplitudes of cytosolic Ca2+ transient and contraction. Furthermore, beta 2AR stimulation did not phosphorylate phospholamban to the same extent as did beta 1AR stimulation. This finding provides a mechanism for the failure of beta 2AR stimulation to accelerate the kinetics of the Ca2+i (cytosolic Ca2+) transient and contraction. These results indicate that the effects of beta 2AR stimulation on Ca2+i transient and contraction are uncoupled from the cAMP production and cAMP-dependent protein phosphorylation and indicate that, in addition to coupling to adenylate cyclase, beta 2AR stimulation also activates other signal transduction pathway(s) to produce changes in cytosolic Ca2+ and contraction.

Actomyosin interaction modulates resting length of unstimulated cardiac ventricular cells.

We sought to determine whether resting or diastolic cardiac myocyte length during low stimulation rates is regulated by myofilament interaction. Cytosolic Ca2+ concentration ([Ca2+]i, via indo 1 fluorescence) and length, in the presence and absence of 2,3-butanedione monoxime (BDM), a potent inhibitor of force production in striated muscle, were measured in rat and guinea pig cardiac myocytes at rest and after electrical stimulation. In tetanized cells BDM reduced steady contraction amplitudes for a given [Ca2+]i. In an actomyosin-sliding filament assay without Ca2+ or regulatory proteins, BDM decreased actin filament velocity along myosin. BDM increased both diastolic and resting cell lengths without changes in [Ca2+]i. The resting cell length also increased when [Ca2+]i was reduced by removing extracellular Ca2+, an effect further enhanced by BDM and by loading cells with the intracellular Ca2+ chelator, 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester. Thus myofilament interaction is present in cardiac cells, both at rest or during low rates of stimulation, and this myofilament interaction is regulated, in part, by the ambient [Ca2+]i.

beta 2-adrenergic receptor-induced p38 MAPK activation is mediated by protein kinase A rather than by Gi or gbeta gamma in adult mouse cardiomyocytes.

Increasing evidence shows that stimulation of beta-adrenergic receptor (AR) activates mitogen-activated protein kinases (MAPKs), in addition to the classical G(s)-adenylyl cyclase-cAMP-dependent protein kinase (PKA) signaling cascade. In the present study, we demonstrate a novel beta(2)-AR-mediated cross-talk between PKA and p38 MAPK in adult mouse cardiac myocytes expressing beta(2)-AR, with a null background of beta(1)beta(2)-AR double knockout. beta(2)-AR stimulation by isoproterenol increased p38 MAPK activity in a time- and dose-dependent manner. Inhibiting G(i) with pertussis toxin or scavenging Gbetagamma with betaARK-ct overexpression could not prevent beta(2)-AR-induced p38 MAPK activation. In contrast, a specific peptide inhibitor of PKA, PKI (5 microm), completely abolished the stimulatory effect of beta(2)-AR, suggesting that beta(2)-AR-induced p38 MAPK activation is mediated via a PKA-dependent mechanism, rather than by G(i) or Gbetagamma. This conclusion was further supported by the ability of forskolin (10 microm), an adenylyl cyclase activator, to elevate p38 MAPK activity in a PKI-sensitive manner. Furthermore, inhibition of p38 MAPK with SB203580 (10 microm) markedly enhanced the beta(2)-AR-mediated contractile response, without altering base-line contractility. These results provide the first evidence that cardiac beta(2)-AR activates p38 MAPK via a PKA-dependent signaling pathway, rather than by G(i) or Gbetagamma, and reveal a novel role of p38 MAPK in regulating cardiac contractility.

Intracellular calcium transients and arrhythmia in isolated heart cells.

Intracellular calcium ([Ca2+]i) elevation may mediate cardiac arrhythmias. However, direct measurement of the rapid alterations of [Ca2+]i on a beat-to-beat basis using fast temporal resolution and without signal averaging in the spontaneously beating in vivo heart is lacking. Furthermore, data from an isolated spontaneously beating myocyte preparation that develops arrhythmia similar to that in the in vivo heart are unavailable. We measured rapid changes of [Ca2+]i with fast temporal resolution in isolated spontaneously beating neonatal rat ventricular myocytes with cell-to-cell communication and characterized the interrelation between [Ca2+]i and arrhythmia. An elevated extracellular calcium ([Ca2+]o) concentration of 10.8 mM induced premature beats, a rapid beating rate (tachyarrhythmia), and chaotic or fibrillatory beating activity in a small group of myocytes. [Ca2+]i levels during systole increased from the nanomolar to micromolar concentration range before arrhythmia development. Spontaneous oscillations of [Ca2+]i during diastole could evoke a spontaneous tachyarrhythmia. In the presence of [Ca2+]i elevation, a spontaneous tachyarrhythmia could induce severe [Ca2+]i overload. Reduction of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid AM (5 microM) in the presence of 10.8 mM [Ca2+]o reversed the arrhythmia. In single ventricular myocytes superfused with 10.8 mM [Ca2+]o, oscillations of membrane potential characteristic of transient inward current occurred that were prevented by ryanodine (0.1 microM), an inhibitor of Ca2+ flux across the sarcoplasmic reticulum. This study characterizes 1) an isolated multicellular myocyte model of arrhythmia similar to that evident in in vivo hearts, 2) elevation of [Ca2+]i with systolic [Ca2+]i levels of 1-3 microM and diastolic [Ca2+]i oscillations before the initiation of arrhythmia, 3) tachyarrhythmia as a cause of severe [Ca2+]i overload, which may be important in the perpetuation and degeneration of arrhythmias, and 4) reversal of arrhythmia with reduction of [Ca2+]i. The results in the isolated myocyte model may have relevance to the generation and perpetuation of certain cardiac arrhythmias associated with calcium overload.

Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation.

1. Single isolated rat cardiac myocytes were loaded with either the pentapotassium salt form or the acetoxymethyl ester (AM) form of the calcium-sensitive fluorescent probe, Indo-1. The relationship of the Indo-1 fluorescence transient, an index of the change in cytosolic calcium [Ca2+]i concentration, to the simultaneously measured cell length during the electrically stimulated twitch originating from slack length at 23 degrees C was evaluated. It was demonstrated that even if the Ca2+ dissociation rate from Indo-1 was assumed to be as slow as 10 s-1, the descending limb ('relaxation phase') of the Indo-1 fluorescence transient induced by excitation under these conditions is in equilibrium with the [Ca2+]i transient. Additionally, the extent of Indo-1 loading employed did not substantially alter the twitch characteristics. 2. A unique relationship between the fluorescence transient and cell length was observed during relaxation of contractions that varied in amplitude. This was manifest as a common trajectory in the cell length vs. [Ca2+]i phase-plane diagrams beginning at the time of cell relengthening. The common trajectory could also be demonstrated in Indo-1 AM-loaded cells. The Indo-1 fluorescence-length relation defined by this common trajectory is steeper than that described by the relation of peak contraction amplitude and peak fluorescence during the twitch contractions. 3. The trajectory of the [Ca2+]i-length relation elicited via an abrupt, rapid, brief (200 ms) pulse of caffeine directly onto the cell surface or by 'tetanization' of cells in the presence of ryanodine is identical to the common [Ca2+]i-length trajectory formed by electrically stimulated contractions of different magnitudes. As the [Ca2+]i and length transients induced by caffeine application or during tetanization in the presence of ryanodine develop with a much slower time course than those elicited by electrical stimulation, the common trajectory is not fortuitous, i.e. it cannot be attributed to equivalent rate-limiting steps for the decrease of [Ca2+]i and cell relengthening. 4. The [Ca2+]i-length relation defined by the common trajectory shifts appropriately in response to perturbations that have previously been demonstrated to alter the steady-state myofilament Ca2+ sensitivity in skinned cardiac fibres. Specifically, the trajectory shifts leftward in response to an acute increase in pH or following the addition of novel myofilament calcium-sensitizing thiadiazinone derivatives; a rightward shift occurs in response to an acute reduction in pH or following the addition of butanedione monoxime.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of spontaneous beta 2-adrenergic activation rescues beta 1-adrenergic contractile response in cardiomyocytes overexpressing beta 2-adrenoceptor.

Cardiac-specific overexpression of the human beta(2)-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand-independent spontaneous beta(2)-AR activation. However, agonist-mediated stimulation of either beta(1)-AR or beta(2)-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of beta(2)-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G(i) proteins in addition to G(s), the contractile response to beta(1)-AR stimulation by norepinephrine and an alpha(1)-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G(i) protein elevation of 1.7-fold in TG4 hearts. Since beta-adrenergic receptor kinase, betaARK1, activity remains unaltered, the unresponsiveness of beta(1)-AR is not caused by betaARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10(-5)m), or a beta(2)-AR inverse agonist, ICI 118,551 (5 x 10(-7)m), to abolish spontaneous beta(2)-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the beta(1)-AR contractile response. The "rescued" contractile response is completely reversed by a beta(1)-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human beta(2)-AR results in elevated base-line cAMP and contraction associated with a marked attenuation of beta(1)-AR response; carbachol pretreatment fully revives the diminished beta(1)-AR contractile response. Thus, we conclude that constitutive beta(2)-AR activation induces a heterologous desensitization of beta(1)-ARs independent of betaARK1 and G(i) proteins; suppression of the constitutive beta(2)-AR signaling by either a beta(2)-AR inverse agonist or stimulation of the muscarinic receptor rescues the beta(1)-ARs from desensitization, permitting agonist-induced contractile response.

Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology.

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.