Jutta Starbatty

Basal and isoprenaline-stimulated cAMP content in failing versus nonfailing human cardiac preparations

We measured force of contraction and cAMP content in human isolated electrically driven right ventricular trabeculae carneae with and without the addition of isoprenaline (0.2 microM). Basal cAMP content was approximately 200% higher in preparations from nonfailing hearts than from hearts with end-stage myocardial failure. Isoprenaline was less effective in increasing force of contraction in failing (by approximately 100%) than in nonfailing cardiac preparations (by approximately 500%). With isoprenaline, cAMP content was approximately 50% lower in failing than in nonfailing preparations. We conclude that the reduced increase in force of contraction of failing human cardiac preparations with isoprenaline added may be causally related to an inadequately increased cAMP content.

Reduced α1- and β2-adrenoceptor-mediated positive inotropic effects in human end-stage heart failure

1 α1‐Adrenoceptor (phenylephrine in the presence of propranolol) and β2‐adrenoceptor (fenoterol)‐mediated positive inotropic effects were investigated in human ventricular preparations isolated from five nonfailing (prospective organ donors) and from eight explanted failing hearts with end‐stage idiopathic dilative cardiomyopathy (NYHA IV). 2 For comparison, the nonselective β‐adrenoceptor agonist isoprenaline, the phosphodiesterase (PDE) inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX), the cardiac glycoside dihydroouabain, and calcium were studied. 3 Furthermore, the influence of IBMX on adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) PDE activity as well as total β‐adrenoceptor density, β1‐ and β2‐adrenoceptor subtype distribution, and α1‐adrenoceptor density were compared in nonfailing and failing human heart preparations. The radioligands (−)‐[125I]‐iodocyanopindolol for β‐adrenoceptor binding and [3H]‐prazosin for α1‐adrenoceptor binding were used. 4 The inotropic responses to calcium and dihydroouabain in failing human hearts were unchanged, whereas the maximal α1‐ and β2‐adrenoceptor‐mediated positive inotropic effects were greatly reduced. The inotropic effects of the other cyclic AMP increasing compounds, i.e. isoprenaline and IBMX, were also reduced to about 60% of the effects observed in nonfailing controls. The potency of these compounds was decreased by factors 4–10. 5 The basal PDE activity and the PDE inhibition by IBMX were similar in nonfailing and failing preparations. 6 The total β‐adrenoceptor density in nonfailing hearts was about 70 fmol mg−1 protein. In failing hearts the total number of β‐adrenoceptors was markedly reduced by about 60%. The β1/β2‐adrenoceptor ratio was shifted from about 80/20% in nonfailing to approximately 60/40% in failing hearts which was due to a selective reduction of β1‐adrenoceptors. The β2‐adrenoceptor population remaining unchanged. α1‐Adrenoceptor density was increased from about 4 fmol mg−1 protein in nonfailing to 10 fmol mg−1 protein in failing hearts. 7 Changes in PDE activity and adrenoceptor downregulation cannot completely explain the reduced positive inotropic effects of α1‐ and β2‐adrenoceptor agonists in failing human hearts. This supports the hypothesis that impairment of other processes such as the coupling between receptor and effector system, i.e. the respective G‐proteins, are equally important in end‐stage heart failure.

Role of guanine nucleotide-binding protein in the regulation by adenosine of cardiac potassium conductance and force of contraction. Evaluation with pertussis toxin

SummaryIn atrial cardiac preparations adenosine exerts a receptor-mediated negative inotropic effect due to an increased potassium conductance. Pretreatment of guinea pigs with pertussis toxin abolished the negative inotropic and action potential shortening effect of adenosine and the adenosine analogue (−)-N6-phenylisopropyladenosine (PIA). As pertussis toxin specifically inactivates guanine nucleotide-binding proteins involved in the signal transfer from receptor binding to specific cell functions, it is concluded that a guanine nucleotide-binding protein is involved in the regulation of the receptor-mediated change in potassium conductance and force of contraction.

Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca2+ sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca2+ sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca2+ transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E′/A′, and higher E/E′ ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca2+ sensitivity, faster Ca2+ transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca2+ sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca2+ transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.

Mechanism underlying the reduced positive inotropic effects of the phosphodiesterase III inhibitors pimobendan, adibendan and saterinone in failing as compared to nonfailing human cardiac muscle preparations.

SummaryThe present study was performed to compare the effects of the new positive inotropic phosphodiesterase III inhibitors pimobendan, adibendan, and saterinone on the isometric force of contraction in electrically driven ventricular trabeculae carneae isolated from explanted failing (end-stage myocardial failure) with those from nonfailing (prospective organ donors) human hearts. In preparations from nonfailing hearts the phosphodiesterase inhibitors, as well as the aβ-adrenoceptor agonist isoprenaline, the cardiac glycoside dihydroouabain, and calcium, which were studied for comparison, revealed pronounced positive inotropic effects. The maximal effects of pimobendan, adibendan, and saterinone amounted to 56%, 36% and 45%, respectively, of the maximal effect of calcium. In contrast, in preparations from failing hearts the phosphodiesterase III inhibitors failed to significantly increase the force of contraction and the effect of isoprenaline was markedly reduced. The effects of dihydroouabain and calcium were almost unaltered. The diminished effects of isoprenaline were restored by the concomitant application of phosphodiesterase inhibitors.To elucidate the underlying mechanism of the lack of effect of the phosphodiesterase III inhibitors in the failing heart we also investigated the inhibitory effects of these compounds on the activities of the phosphodiesterase isoenzymes I–III separated by DEAE-cellulose chromatography from both kinds of myocardial tissue. Furthermore, the effects of pimobendan and isoprenaline on the content of cyclic adenosine monophosphate (determined by radioimmunoassays) of intact contracting trabeculae were studied. The lack of effect of the phosphodiesterase inhibitors in failing human hearts could not be explained by an altered phosphodiesterase inhibition, since the properties of the phosphodiesterase isoenzymes I–III and also the inhibitory effects of the phosphodiesterase inhibitors on these isoenzymes did not differ between failing and nonfailing human myocardial tissue. Instead, it may be due to a diminished formation of cyclic adenosine monophosphate in failing hearts, presumably caused mainly by a defect in receptor-adenylate cyclase coupling at least in idiopathic dilated cardiomyopathy. Both the basal and the pimobendan-stimulated or isoprenaline-stimulated contents of cyclic adenosine monophosphate of intact contracting trabeculae from failing hearts were decreased compared with the levels in nonfailing hearts. However, under the combined action of isoprenaline and pimobendan the cyclic adenosine monophosphate level reached values as high as with each compound alone in nonfailing preparations, and in addition the positive inotropic effect of isoprenaline was restored.These findings may have important clinical implications. Along with the elevated levels of circulating catecholamines the positive inotropic effects of the phosphodiesterase inhibitors may be maintained in patients with heart failure. Furthermore, the concomitant application of a β-adrenoceptor agonist and a phosphodiesterase inhibitor might be beneficial in terminal heart failure refractory to conventional therapeutic regimens.

Cardiac repair in guinea pigs with human engineered heart tissue from induced pluripotent stem cells

Human engineered heart tissue derived from induced pluripotent stem cells improves cardiac function in guinea pigs. A patch for a broken heart A heart attack destroys cardiac muscle, resulting in a fibrotic scar. Weinberger et al. created a living patch for injured hearts using endothelial and cardiac cells grown from human induced pluripotent stem cells. These three-dimensional strips were placed over injured areas of guinea pig hearts; 28 days later, the injured area was partly remuscularized, and the heart pumped ~30% better than just after the injury. The grafts also contained new blood vessels and, in some cases, were electrically coupled to the healthy parts of the heart. These human heart patches may one day help patients recover cardiac function after a heart attack. Myocardial injury results in a loss of contractile tissue mass that, in the absence of efficient regeneration, is essentially irreversible. Transplantation of human pluripotent stem cell–derived cardiomyocytes has beneficial but variable effects. We created human engineered heart tissue (hEHT) strips from human induced pluripotent stem cell (hiPSC)–derived cardiomyocytes and hiPSC-derived endothelial cells. The hEHTs were transplanted onto large defects (22% of the left ventricular wall, 35% decline in left ventricular function) of guinea pig hearts 7 days after cryoinjury, and the results were compared with those obtained with human endothelial cell patches (hEETs) or cell-free patches. Twenty-eight days after transplantation, the hearts repaired with hEHT strips exhibited, within the scar, human heart muscle grafts, which had remuscularized 12% of the infarct area. These grafts showed cardiomyocyte proliferation, vascularization, and evidence for electrical coupling to the intact heart tissue in a subset of engrafted hearts. hEHT strips improved left ventricular function by 31% compared to that before implantation, whereas the hEET or cell-free patches had no effect. Together, our study demonstrates that three-dimensional human heart muscle constructs can repair the injured heart.

Evidence for adenosine receptor-mediated isoprenaline-antagonistic effects of the adenosine analogs PIA and NECA on force of contraction in guinea-pig atrial and ventricular cardiac preparations

SummaryThe effects of the adenosine agonists (−)-N6-phenylisopropyladenosine (PIA) and 5′-N-ethylcarboxamideadenosine (NECA) on force of contraction, adenylate cyclase activity and normal as well as slow action potentials were studied in guinea-pig isolatedatrial (left auricles) andventricular preparations (papillary muscles).Inauricles PIA and NECA exerted concentration-dependent negative inotropic effects with similar potenticies (mean EC50:0.05 μmol l−1 for PIA and 0.03 μmol l−1 for NECA). Similar results were obtained in the presence of isoprenaline.Inpapillary muscles PIA and NECA alone had no effect on force of contraction but produced negative inotropic effects in the presence of isoprenaline (mean EC50:0.19 μmol l−1 for PIA and 0.10 μmol l−1 for NECA).In both preparations, the negative inotropic effects of PIA and NECA in the presence of isoprenaline were antagonized by the adenosine receptor antagonist 8-phenyltheophylline.In both preparations, PIA and NECA did not affect adenylate cyclase activity, both in the absence and presence of isoprenaline.Inauricles the negative inotropic effects of both nucleosides were accompanied by shortening of the action potential. This effect was also observed in the presence of isoprenaline. Inpapillary muscles the adenosine analogs did not detectably alter the shape of the normal action potential. Ca2+-dependent slow action potentials elicited in potassium-depolarized preparations also remained unaltered in the presence of PIA or NECA alone. However, the isoprenaline-induced enhancement of the maximal rate of depolarization of slow action potentials was attenuated by PIA or NECA.It is concluded that in guinea-pig atrial and ventricular cardiac preparations the adenosine analogs PIA and NECA exert isoprenaline-antagonistic effects on force of contraction via adenosine receptors the existence of which can thus be shown in a functional way. These receptors are not detectably coupled to the adenylate cyclase. The negative inotropic effect in theauricle is most likely due to a shortening of the action potential resulting from an activation of potassium channels, which in turn indirectly reduces the Ca2+ influx during the action potential. In theventricle the adenosine receptor is either not linked to these potassium channels or adenosine-sensitive potassium channels do not exist in the ventricle. Instead the activation of the receptor causes a decrease of the slow Ca2+ inward current but this effect is observed only when the slow Ca2+ inward current had previously been enhanced by a cyclic AMP-dependent mechanism.

Effects of isomazole on force of contraction and phosphodiesterase isoenzymes I-IV in nonfailing and failing human hearts.

The phosphodiesterase (PDE) inhibitor isomazole increased the force of contraction to 278.3 ± 89.1% (n = 7) of the predrug value in ventricular trabeculae carneae isolated from nonfailing human hearts. This effect can be attributed mainly to a PDE III or a combined PDE III/IV inhibition since at the concentration of the maximal positive inotropic effect of isomazole, PDE III and PDE IV were completely inhibited. In explanted failing human hearts (end-stage myocardial failure, NYHA IV), isomazole increased the force of contraction only marginally to 110.1 ± 10.7% of the predrug value. The lack of a distinct positive inotropic efficacy of isomazole in failing human hearts could not be explained by an impairment of PDE inhibition since the properties of the PDE I-IV isoenzymes separated by DEAE-Sepharose chromatography and the inhibitory effects of isomazole did not differ in both preparations. The positive inotropic effect of the β-adrenoceptor agonist isoprenaline was also reduced in failing hearts. However, in the presence of isomazole, the diminished positive inotropic effect of isoprenaline was restored to values obtained with isoprenaline alone in nonfailing hearts. Thus, the decreased effect of inotropic drugs like isoprenaline or isomazole in preparations from failing human heart might be explained mainly by a diminished cAMP formation due to a defect in receptor-adenylate cyclase coupling.

Cerebral expression of the α2-subunit of soluble guanylyl cyclase is linked to cerebral maturation and sensory pathway refinement during postnatal development

Abstract Soluble guanylyl cylase (sGC) has been identified for being a receptor for the gaseous transmitters nitric oxide and cabon monoxide. Currently four subunits α 1 , α 2 , β 1 , and β 2 have been characterized. Heterodimers of α and β-subunits as well as homodimers of the β 2 -subunit are known to constitute functional sGC which use GTP to form cGMP a potent signal molecule in a multitude of second messenger cascades. Since NO-cGMP signaling plays a pivotal role in neuronal development we analyzed the maturational expression pattern of the newly characterized α 2 -subunit of sGC within the brain of Wistar rats by means of RNase protection assay and immunohistochemistry. α 2 -subunit mRNA as well as immunoreactive α 2 -protein increased during postnatal cerebral development. Topographical analysis revealed a selective high expression of the α 2 -subunit in the choroid plexus and within developing sensory systems involving the olfactory and somatosensory system of the forebrain as well as parts of the auditory and visual system within the hindbrain. In cultured cortical neurons the α 2 -subunit was localized to the cell membrane, especially along neuronal processes. During the first 11 days of postnatal development several cerebral regions showed a distinct expression of the α 2 -subunit which was not paralleled by the α 1 /β 1 -subunits especially within the developing thalamo-cortical circuitries of the somatosensory system. However, at later developmental stages all three subunits became more homogenously distributed among most cerebral regions, indicating that functional α 1 /β 1 and α 2/ β 1 heterodimers of sGC could be formed. Our findings indicate that the α 2 -subunit is an essential developmentally regulated constituent of cerebral sensory systems during maturation. In addition the α 2 -subunit may serve other functions than forming a functional heterodimer of sGC during the early phases of sensory pathway refinement.

The β2 subunit of nitric oxide-sensitive guanylyl cyclase is developmentally regulated in rat kidney

Abstract. We have recently shown that nitric oxide activates the β2 subunit of soluble guanylyl cyclase. In the present study, we show developmental regulation of this subunit. Analysis of mRNA expression by RT-PCR and RNase protection analysis in kidneys revealed no expression of the β2 subunit in neonatal and strong expression in adult rats. A reciprocal regulation with much lower expression levels was observed in rat lung. Further examination of kidneys from 3, 6, 16, 22, 25, 31 and 36-day-old rats showed that significant expression appears between postnatal day 16 and 22. Isolation of the rat β2 promoter by genome walking and cloning into a reporter gene vector showed promoter activity for the sense but not the antisense construct providing an in vitro assay for further analysis of the developmental β2 subunit regulation.