Mechanistic role of the CREB-regulated transcription coactivator 1 in cardiac hypertrophy.

The sympathetic nervous system is the main stimulator of cardiac function. While acute activation of the ß-adrenoceptors exerts positive inotropic and lusitropic effects by increasing cAMP and Ca2+, chronically enhanced sympathetic tone with changed ß-adrenergic signaling leads to alterations of gene expression and remodeling. The CREB-regulated transcription coactivator 1 (CRTC1) is activated by cAMP and Ca2+. In the present study, the regulation of CRTC1 in cardiomyocytes and its effect on cardiac function and growth was investigated. In cardiomyocytes, isoprenaline induced dephosphorylation, and thus activation of CRTC1, which was prevented by propranolol. Crtc1-deficient mice exhibited left ventricular dysfunction, hypertrophy and enlarged cardiomyocytes. However, isoprenaline-induced contractility of isolated trabeculae or phosphorylation of cardiac troponin I, cardiac myosin-binding protein C, phospholamban, and ryanodine receptor were not altered, suggesting that cardiac dysfunction was due to the global lack of Crtc1. The mRNA and protein levels of the Gαq GTPase activating protein regulator of G-protein signaling 2 (RGS2) were lower in hearts of Crtc1-deficient mice. Chromatin immunoprecipitation and reporter gene assays showed stimulation of the Rgs2 promoter by CRTC1. In Crtc1-deficient cardiomyocytes, phosphorylation of the Gαq-downstream kinase ERK was enhanced. CRTC1 content was higher in cardiac tissue from patients with aortic stenosis or hypertrophic cardiomyopathy and from two murine models mimicking these diseases. These data suggest that increased CRTC1 in maladaptive hypertrophy presents a compensatory mechanism to delay disease progression in part by enhancing Rgs2 gene transcription. Furthermore, the present study demonstrates an important role of CRTC1 in the regulation of cardiac function and growth.

AkrinorTM, a Cafedrine/ Theodrenaline Mixture (20:1), Increases Force of Contraction of Human Atrial Myocardium But Does Not Constrict Internal Mammary Artery In Vitro.

Background: Intraoperative hypotension is a common problem and direct or indirect sympathomimetic drugs are frequently needed to stabilize blood pressure. AkrinorTM consists of the direct and the indirect sympathomimetic noradrenaline and norephedrine. Both substances are covalently bound to the phosphodiesterase (PDE) inhibitor theophylline, yielding theodrenaline and cafedrine, respectively. We investigated pharmacodynamic effects of AkrinorTM and its constituents on contractile force and tension in human atrial trabeculae and internal A. mammaria rings. Methods: Isometric contractions were measured in human atrial trabeculae at 1 Hz and 37°C. CGP 20712A and ICI 118,551 were used to elaborate ß1- and ß2-adrenoceptor (AR) subtypes involved and phenoxybenzamine to estimate indirect sympathomimetic action. PDE-inhibition was measured as a potentiation of force increase upon direct activation of adenylyl cyclase by forskolin. Human A. mammaria preparations were used to estimate intrinsic vasoconstriction and impact on the noradrenaline-induced vasoconstriction. Results: Clinically relevant concentrations of AkrinorTM (4.2-420 mg/l) robustly increased force in human atrial trabeculae (EC50 41 ± 3 mg/l). This direct sympathomimetic action was mediated via ß1-AR and the effect size was as large as with high concentrations of calcium. Only the highest and clinically irrelevant concentration of AkrinorTM increased the potency of forskolin to a minor extent. Norephedrine has lost its indirect sympathomimetic effect when bound to theophylline. Increasing concentrations of AkrinorTM (4.2-168 mg/l) alone did not affect the tension of human A. mammaria interna rings, but shifted the noradrenaline curve rightward from -logEC50 6.18 ± 0.08 to 5.23 ± 0.05 M. Conclusion: AkrinorTM increased cardiac contractile force by direct sympathomimetic actions and PDE inhibition, did not constrict A. mammaria preparations, but shifted the concentration-response curve to the right, compatible with an α-AR antagonistic effect or PDE inhibition. The pharmacodynamic profile and potency of AkrinorTM differs from noradrenaline and norephedrine in vitro. We anticipate metabolism of theodrenaline and cafedrine resulting in a different pharmacodynamic profile of AkrinorTMin vivo.

Blinded Contractility Analysis in hiPSC-Cardiomyocytes in Engineered Heart Tissue Format: Comparison With Human Atrial Trabeculae.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) may serve as a new assay for drug testing in a human context, but their validity particularly for the evaluation of inotropic drug effects remains unclear. In this blinded analysis, we compared the effects of 10 indicator compounds with known inotropic effects in electrically stimulated (1.5 Hz) hiPSC-CM-derived 3-dimensional engineered heart tissue (EHT) and human atrial trabeculae (hAT). Human EHTs were prepared from iCell hiPSC-CM, hAT obtained at routine heart surgery. Mean intra-batch variation coefficient in baseline force measurement was 17% for EHT and 49% for hAT. The PDE-inhibitor milrinone did not affect EHT contraction force, but increased force in hAT. Citalopram (selective serotonin reuptake inhibitor), nifedipine (LTCC-blocker) and lidocaine (Na+ channel-blocker) had negative inotropic effects on EHT and hAT. Formoterol (beta-2 agonist) had positive lusitropic but no inotropic effect in EHT, and positive clinotropic, lusitropic, and inotropic effects in hAT. Tacrolimus (calcineurin-inhibitor) had a negative inotropic effect in EHTs, but no effect in hAT. Digoxin (Na+-K+-ATPase-inhibitor) showed a positive inotropic effect only in EHTs, but no effect in hAT probably due to short incubation time. Ryanodine (ryanodine receptor-inhibitor) reduced contraction force in both models. Rolipram and acetylsalicylic acid showed noninterpretable results in hAT. Contraction amplitude and kinetics were more stable over time and less variable in hiPSC-EHTs than hAT. HiPSC-EHT faithfully detected cAMP-dependent and -independent positive and negative inotropic effects, but limited beta-2 adrenergic or PDE3 effects, compatible with an immature CM phenotype.

Ranolazine antagonizes catecholamine-induced dysfunction in isolated cardiomyocytes, but lacks long-term therapeutic effects in vivo in a mouse model of hypertrophic cardiomyopathy.

AIMS: Hypertrophic cardiomyopathy (HCM) is often accompanied by increased myofilament Ca(2+) sensitivity and diastolic dysfunction. Recent findings indicate increased late Na(+) current density in human HCM cardiomyocytes. Since ranolazine has the potential to decrease myofilament Ca(2+) sensitivity and late Na(+) current, we investigated its effects in an Mybpc3-targeted knock-in (KI) mouse model of HCM. METHODS AND RESULTS: Unloaded sarcomere shortening and Ca(2+) transients were measured in KI and wild-type (WT) cardiomyocytes. Measurements were performed at baseline (1 Hz) and under increased workload (30 nM isoprenaline (ISO), 5 Hz) in the absence or presence of 10 µM ranolazine. KI myocytes showed shorter diastolic sarcomere length at baseline, stronger inotropic response to ISO, and drastic drop of diastolic sarcomere length under increased workload. Ranolazine attenuated ISO responses in WT and KI cells and prevented workload-induced diastolic failure in KI. Late Na(+) current density was diminished and insensitive to ranolazine in KI cardiomyocytes. Ca(2+) sensitivity of skinned KI trabeculae was slightly decreased by ranolazine. Phosphorylation analysis of cAMP-dependent protein kinase A-target proteins and ISO concentration-response measurements on muscle strips indicated antagonism at ß-adrenoceptors with 10 µM ranolazine shifting the ISO response by 0.6 log units. Six-month treatment with ranolazine (plasma level >20 µM) demonstrated a ß-blocking effect, but did not reverse cardiac hypertrophy or dysfunction in KI mice. CONCLUSION: Ranolazine improved tolerance to high workload in mouse HCM cardiomyocytes, not by blocking late Na(+) current, but by antagonizing ß-adrenergic stimulation and slightly desensitizing myofilaments to Ca(2+). This effect did not translate in therapeutic efficacy in vivo.

ß 1 Adrenoceptor antagonistic effects of the supposedly selective ß 2 adrenoceptor antagonist ICI 118,551 on the positive inotropic effect of adrenaline in murine hearts.

Studies on the relative contribution of ß 1- and ß 2-adrenoceptors (AR) generally employ selective ß 1- and ß 2-AR antagonists such as CGP 20712A and ICI 118,551, respectively, and assume that antagonism by one of these compounds indicates mediation by the respective AR subtype. Here, we evaluated the ß 2-AR-selectivity of ICI 118,551 in ventricular muscle strips of transgenic mice lacking ß 1-AR (ß 1-KO), ß 2-AR (ß 2-KO), or both (ß 1/ß 2-KO). Strips were electrically driven and force development was measured. In wild type (WT), ICI 118,551 (100 nmol/L) shifted the concentration-response curve (CRC) for adrenaline by about 0.5 log units to the right, corresponding to the known affinity of ICI 118,551 to ß 1-AR but not to ß 2-AR. Conversely, the phosphodiesterase inhibitor rolipram (10 µmol/L) shifted the CRC to the left, but did not enlarge the ICI 118,551 shift, indicating exclusive ß 1-AR mediation even when PDE4 is inactive. In line with this, rolipram and ICI 118,551 had similar effects in ß 2-KO than in WT. In contrast, ß 1-KO did not show any inotropic reaction to adrenaline (+/- rolipram). In WT, the ß 1-AR selective antagonist CGP 20712A (100 nmol/L) shifted the CRC for isoprenaline by 2.1 log units, corresponding to the affinity of CGP 20712A to ß 1-AR. Rolipram increased the sensitivity to adrenaline independently of the presence of CGP 20712A. We conclude that effects sensitive to the ß 2-AR antagonist ICI 118,551 are not necessarily ß 2-AR-mediated and CGP 20712A-resistant effects cannot be simply interpreted as ß 2-AR-mediated. Catecholamine effects in murine ventricles strictly depend on ß 1-AR, even if PDE 4 is blocked.

Prostaglandin E2 does not attenuate adrenergic-induced cardiac contractile response.

Systemic inflammation may contribute to heart failure. PGE2 was recently suggested to mediate inflammation-induced impairment of cardiac function by desensitizing the murine heart to isoprenaline. Given the magnitude of the reported effect and the potential relevance, we sought to reproduce it in the human heart. Human trabeculae were prepared from the right atrial tissue obtained during heart surgery and from the right ventricle of two explanted human failing hearts. Muscle strips were electrically driven and isometric force development was measured. PGE2 was given at a single concentration (0.1 µM). Norepinephrine was used to activate ß1-adrenoceptors, epinephrine to activate ß2-adrenoceptors in atrial trabeculae. Isoprenaline was used in ventricular tissue. All patients were in sinus rhythm. Murine ventricular strips were used for comparison and stimulated with isoprenaline. The pharmacological activity of the PGE2 batch was confirmed by assessing concentration-dependent vasoconstriction in murine aorta. We used atrial and ventricular trabeculae from humans. Exposure to PGE2 (15 min) did not affect contractility when compared to time-matched controls. PGE2 neither altered the sensitivity or efficacy of ß1- or ß2-adrenoceptor-mediated stimulation of force in human atrial or in ventricular trabeculae for nonselective ß1- or ß2-adrenoceptor-stimulation. Surprisingly, PGE2 also did not affect -logEC50 values or maximum catecholamine-stimulated force in ventricular strips from mice, whereas it induced vasoconstriction in aortic rings with an -logEC50 of 5.0 (n = 6). Our data do not support a role for PGE2 in regulating catecholamine inotropy, neither in mice nor in humans.