okadaic-acid and Heart-Failure

L-type Ca(2+) channels (LTCC) play a crucial role in cardiac excitation-contraction coupling. We previously found that in failing ventricular myocytes of mice chronically treated with isoproterenol, basal t-tubular (TT) LTCC activity was halved by activation of protein phosphatase (PP)2A whereas basal surface sarcolemmal (SS) LTCC activity was doubled by inhibition of PP1. Interestingly, chronic treatment of these mice with pertussis toxin almost completely normalized TT and SS LTCC densities and cardiac contractility. In the present study, we therefore sought to identify the Gi/o protein-coupled receptors in cardiac myocytes (i.e. β2-adrenergic, M2-muscarinic and A1-adenosine receptors) that are responsible for these abnormalities in heart failure by chronically administrating mice a selective antagonist of each receptor (ICI118,551, atropine and 8-cyclopentyl-1,3-dipropilxanthine (DPCPX), respectively) with isoproterenol. Compared with mice treated with isoproterenol alone, mice treated with isoproterenol plus ICI118,551 or atropine, but not DPCPX showed significantly lower lung weight/tibial length, higher fractional shortening, lower left ventricular end-diastolic pressure and higher dP/dtmax and dP/dtmin. In addition, ventricular myocytes of mice treated with isoproterenol plus ICI118,551 or atropine, but not DPCPX exhibited significantly higher TT and lower SS LTCC current densities than those of mice treated with isoproterenol alone due to normalization of the PP activities. These results indicate that β2-adrenergic, M2-muscarinic, but not A1-adenosine receptors contribute to reduced ventricular contractility at least partially by decreasing basal TT LTCC activity in heart failure. Therefore, antagonists of β2-adrenergic and/or M2-muscarinic receptors can be good adjuncts to β1-adrenergic receptor antagonists in the treatment of heart failure.

Topics: Adenosine A1 Receptor Antagonists; Adrenergic beta-Antagonists; Animals; Atropine; Calcium Channels, L-Type; Enzyme Inhibitors; Heart Failure; Male; Mice; Mice, Inbred C57BL; Muscarinic Antagonists; Myocardial Contraction; Okadaic Acid; Phosphoprotein Phosphatases; Propanolamines; Receptor, Muscarinic M2; Receptors, Adrenergic, beta-2; Ventricular Function, Left; Xanthines

Cardiac L-type Ca(2+)-currents show distinct alterations in chronic heart failure, including increased single-channel activity and blunted adrenergic stimulation, but minor changes of whole-cell currents. Expression of L-type Ca(2+)-channel beta(2)-subunits is enhanced in human failing hearts. In order to determine whether prolonged alteration of Ca(2+)-channel gating by beta(2)-subunits contributes to heart failure pathogenesis, we generated and characterized transgenic mice with cardiac overexpression of a beta(2a)-subunit or the pore Ca(v)1.2 or both, respectively.. Four weeks induction of cardiac-specific overexpression of rat beta(2a)-subunits shifted steady-state activation and inactivation of whole-cell currents towards more negative potentials, leading to increased Ca(2+)-current density at more negative test potentials. Activity of single Ca(2+)-channels was increased in myocytes isolated from beta(2a)-transgenic mice. Ca(2+)-current stimulation by 8-Br-cAMP and okadaic acid was blunted in beta(2a)-transgenic myocytes. In vivo investigation revealed hypotension and bradycardia upon Ca(v)1.2-transgene expression but not in mice only overexpressing beta(2a). Double-transgenics showed cardiac arrhythmia. Interstitial fibrosis was aggravated by the beta(2a)-transgene compared with Ca(v)1.2-transgene expression alone. Overt cardiac hypertrophy was not observed in any model.. Cardiac overexpression of a Ca(2+)-channel beta(2a)-subunit alone is sufficient to induce Ca(2+)-channel properties characteristic of chronic human heart failure. beta(2a)-overexpression by itself did not induce cardiac hypertrophy or contractile dysfunction, but aggravated the development of arrhythmia and fibrosis in Ca(v)1.2-transgenic mice.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Arrhythmias, Cardiac; Calcium Channels, L-Type; Chronic Disease; Disease Models, Animal; Fibrosis; Heart Failure; Heart Rate; Humans; Mice; Mice, Transgenic; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Okadaic Acid; Patch-Clamp Techniques; Protein Subunits

The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by beta-adrenergic receptor (beta-AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni2+-sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF (P<0.05 versus control). Despite this modest increase in protein, the basal peak outward NCX current was increased almost 5-fold in HF (P<0.05 versus control). Stimulation with isoproterenol, however, increased the control currents to a significantly greater extent than HF (500% increase in control versus 100% increase in HF, P<0.01); peak stimulated current was not different in HF and control. This reduction in responsiveness to beta-AR stimulation was refractory to forskolin, 8-Br-cAMP, or okadaic acid stimulation. In vitro protein kinase A back-phosphorylation revealed higher phosphorylation capacity of NCX protein in control versus HF, consistent with increased phosphorylation in vivo (hyperphosphorylation) in HF. Protein phosphatase type 1 exposure resulted in a significant reduction (73%) in peak basal current in HF (compared with no significant difference in controls), confirming that the increased basal NCX current in HF is predominantly attributable to hyperphosphorylation. NCX expression and activity are thus increased in HF, although beta-AR responsiveness is decreased because of NCX hyperphosphorylation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenergic beta-Agonists; Animals; Cells, Cultured; Colforsin; Cyclic AMP-Dependent Protein Kinases; Female; Heart Failure; Isoproterenol; Male; Membrane Potentials; Myocardium; Myocytes, Cardiac; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Receptors, Adrenergic, beta; Sarcolemma; Sodium-Calcium Exchanger; Swine

Ca2+ influx through the L-type calcium channel (LTCC) induces Ca2+ release from the sarcoplasmic reticulum (SR) and maintains SR Ca2+ loading. Alterations in LTCC properties, their contribution to the blunted adrenergic responsiveness in failing hearts and their recovery after support with LV assist devices (LVAD) were studied. L-type Ca2+ current (I(Ca,L)) was measured under basal conditions and in the presence of isoproterenol (ISO), dibutyryl-cAMP (db-cAMP), Bay K 8644 (BayK), Okadaic acid (OA, a phosphatase inhibitor), and phosphatase 2A (PP2A) in nonfailing (NF), failing (F), and LVAD-supported human left ventricular myocytes (HVMs). Basal I(Ca,L) density was not different in the 3 groups but I(Ca,L) was activated at more negative voltages in F- and LVAD- versus NF-HVMs (V(0.5): -7.18+/-1.4 and -7.0+/-0.9 versus 0.46+/-1.1 mV). Both ISO and db-cAMP increased I(Ca,L) in NF- and LVAD- significantly more than in F-HVMs (NF >LVAD> F: ISO: 90+/-15% versus 77+/-19% versus 24+/-12%; db-cAMP: 235%>172%>90%). ISO caused a significant leftward shift of the I(Ca,L) activation curve in NF- and LVAD- but not in F-HVMs. After ISO and db-cAMP, the I(Ca,L) activation was not significantly different between groups. BayK also increased I(Ca,L) more in NF- (81+/-30%) and LVAD- (70+/-15%) than in F- (51+/-8%) HVMs. OA increased I(Ca, L) by 85.6% in NF-HVMs but had no effect in F-HVMs, while PP2A decreased I(Ca, L) in F-HVMs by 35% but had no effect in NF-HVMs. These results suggest that the density of LTCC is reduced in F-HVMs but basal I(Ca,L) density is maintained by increasing in LTCC phosphorylation.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenergic beta-Agonists; Bucladesine; Calcium Channel Agonists; Calcium Channels, L-Type; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Heart Failure; Heart Ventricles; Heart-Assist Devices; Humans; Isoproterenol; Membrane Potentials; Middle Aged; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2

Topics: Adrenergic beta-Agonists; Animals; Disease Models, Animal; Enzyme Inhibitors; Heart Failure; Heart Ventricles; Isoproterenol; Muscle Cells; Okadaic Acid; Sodium-Calcium Exchanger; Swine

Downregulation of the L-type Ca(2+) current (I(Ca)) is an important determinant of the electrical remodeling of diseased atria. Using a rat model of heart failure (HF) due to ischemic cardiopathy, we studied I(Ca) in isolated left atrial myocytes with the whole-cell patch-clamp technique and biochemical assays. I(Ca) density was markedly reduced (1.7+/-0.1 pA/pF) compared with sham-operated rats (S) (4.1+/-0.2 pA/pF), but its gating properties were unchanged. Calcium channel alpha(1C)-subunit quantities were not significantly different between S and HF. The beta-adrenergic agonist isoproterenol (1 micromol/L) had far greater stimulatory effects on I(Ca) in HF than in S (2.5- versus 1-fold), thereby suppressing the difference in current density. Dialyzing cells with 100 micromol/L cAMP or pretreating them with the phosphatase inhibitor okadaic acid also increased I(Ca) and suppressed the difference in density between S and HF. Intracellular cAMP content was reduced more in HF than in S. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine had a greater effect on I(Ca) in HF than in S (76.0+/-11.2% versus 15.8+/-21.2%), whereas the inhibitory effect of atrial natriuretic peptide on I(Ca) was more important in S than in HF (54.1+/-4.8% versus 24.3+/-8.8%). Cyclic GMP extruded from HF myocytes was enhanced compared with S (55.8+/-8.0 versus 6.2+/-4.0 pmol. mL(-1)). Thus, I(Ca) downregulation in atrial myocytes from rats with heart failure is caused by changes in basal cAMP-dependent regulation of the current and is associated with increased response to catecholamines.

Topics: 1-Methyl-3-isobutylxanthine; Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Calcium; Calcium Channels, L-Type; Catecholamines; Cell Separation; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Down-Regulation; Enzyme Inhibitors; Heart Atria; Heart Failure; Male; Myocardial Infarction; Myocardium; Okadaic Acid; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Rats; Rats, Wistar; Signal Transduction