Elizabeth Kelso

Temporal characteristics of cardiomyocyte hypertrophy in the spontaneously hypertensive rat.

BACKGROUND The spontaneously hypertensive rat (SHR) is frequently used as model of cardiovascular disease, with considerable disparity in reported parameters of hypertrophy. The aim of this study was to assess the temporal changes occurring during the development and progression of cardiomyocyte hypertrophy in SHR, subsequent to pressure overload, compared to changes associated with normal aging using the normotensive Wistar-Kyoto (WKY) rat. METHODS Ventricular cardiomyocytes were isolated from rats at 8, 12, 16, 20 and 24 weeks, and parameters of hypertrophy (cell dimensions, protein mass, de novo protein synthesis, and gene expression) and function (contraction and hypertrophic responsiveness in vitro) were assessed. RESULTS Hypertension was evident at > or =7 weeks in SHRs. Heart:body mass ratio, cardiomyocyte protein mass and width were elevated (P<.05) in SHRs at 16-20 weeks compared to WKYs. In SHRs compared to WKYs at 16 weeks, there was a transient increase (P<.05) in protein synthesis, enhanced hypertrophic responsiveness to phorbol-12-myristate-13-acetate, and induced hypertrophic responsiveness to isoprenaline. Skeletal-alpha-actin mRNA was detected in SHR but not WKY cells at all ages. ANP mRNA was lower in SHR than in WKY cells at 8-20, but progressively increased (P<.05) from 12 to 24 weeks within SHRs. Contractile function increased (P<.05) at 20 weeks in SHR compared to WKY rats. CONCLUSION Structural and functional changes occurring at the cellular level in the myocardium of SHR follow a distinct pattern, such that pressure overload was initially accompanied by expressional changes (8-12 weeks), followed by active hypertrophic growth and enhanced function (16-20 weeks), which subsequently decelerated as stable compensation was attained.

Alterations in vascular matrix metalloproteinase due to ageing and chronic hypertension: effects of endothelin receptor blockade.

Objective To determine the effects of age and dual endothelin (ET)A/ETB receptor antagonism (bosentan) on aortic matrix metalloproteinase (MMP) abundance and tissue inhibitor of metalloproteinase (TIMP) expression in normotensive Wistar–Kyoto (WKY) and spontaneously hypertensive rats (SHR). Methods Male SHR and control WKY rats were randomly assigned to receive placebo or bosentan (100 mg/kg per day) for 3 months. Animals were killed under terminal anaesthesia at either 20 weeks (adult) or 17–20 months (senescent). Aortic gelatinase activity was determined by zymography, whereas MT-1 MMP and TIMP-1 expression were assessed by immunoblotting. Results In WKY rats, aortic MMP-2 but not proMMP-2 activity was 3.6-fold higher (P < 0.02) in the senescent compared with the adult group. TIMP-1 (twofold) and MT-1 MMP (3.8-fold) expression increased (P < 0.05) with age in the WKY groups. Short-term hypertension (adult SHR versus adult WKY) increased MMP-2 to 74.7 ± 14.1 from 18.9 ± 3.5 arbitrary units (AU) (P = 0.0012), but did not alter proMMP-2 activity. This increased further on progression to chronic hypertension (117.4 ± 12.2 versus 74.7 ± 14.1 AU; P < 0.02). Bosentan decreased MMP-2 (78.9 ± 3.8 versus 117.4 ± 12.2 AU; P = 0.014) and proMMP-2 activity (P < 0.006) in the senescent SHR group. Conclusion Ageing and the development/progression of hypertension are associated with increased MMP-2 activity in the aorta, which is consistent with ongoing remodelling of the vasculature. However, the underlying mechanisms regulating MMP-2 abundance in ageing and hypertension appear to be divergent, as MT-1 MMP expression is differentially altered. Dual ETA/ETB receptor antagonism did not alter the age-dependent increase in aortic MMP activity in normotensive rats. However, bosentan decreased pro and active MMP-2 activity in senescent SHR rats, indicating that ET modulates late events in vascular remodelling in hypertension.

Activation of immune and inflammatory systems in chronic heart failure: novel therapeutic approaches

Despite extensive research and novel treatments, chronic heart failure (CHF) remains a cause of high morbidity and mortality. Mounting evidence suggested that immune activation and inflammation play critical roles in the pathogenesis of CHF. In this review, we examine the current evidence regarding this contemporary pathophysiological mechanism, and evaluate the effects of conventional and novel cardiovascular drugs, such as calcium sensitisers and statins, on the immune and inflammatory mediators network. Although therapies, which specifically antagonise tumour necrosis factor‐α have not demonstrated considerable benefit in patients with CHF, there is an increasing evidence to suggest greater value from non‐specific anti‐inflammatory approaches, including: pentoxifylline, intravenous immunoglobulin, immune modulation therapy, growth hormones, physical training and nutrition regulation. Several innovative therapeutic targets, such as peroxisome proliferator‐activated receptor γ activators, Rho‐kinase, p38 mitogen‐activated protein kinase, nuclear transcription factor NF‐κB, recovering or augmenting parasympathetic tone, cardiac resynchronisation therapy, macrophage inhibitors and chemokine receptor antagonists, are briefly discussed in this review. While we have recently demonstrated the potential merits of combining low‐dose methotrexate with conventional therapy, through extensively modulating the activated immune and inflammatory mediators network, there is a need for further rigorous research of this complex network, especially involving current promising therapies which modulate this system. Such evidence has the potential to revolutionise changes for the management of this disorder. Based on the ‘heterogeneity’ of immune activation and inflammation among different CHF populations, an ‘optimised combination treatment’ may offer exciting benefits for individual therapy in the future.

Involvement of endothelin (ET)A and ETB receptors in the hypertrophic effects of ET-1 in rabbit ventricular cardiomyocytes.

The question was addressed whether endothelin-1 (ET-1) exerts hypertrophic effects in cardiomyocytes isolated from ventricles of adult rabbits and maintained in short-term (24 h) serum-free primary culture providing mechanical quiescence. ET-1 (> or =100 pM) increased significantly total mass of cellular protein and incorporation of L-U-[(14)C]phenylalanine and 2-[(14)C]uridine into cellular protein and RNA, respectively. Cycloheximide (35 microM), an inhibitor of protein synthesis, significantly reduced the incorporation of L-U-[(14)C]phenylalanine and 2-[(14)C]uridine into cellular protein and RNA, respectively, under control conditions and in response to ET-1. Actinomycin D (5 microM), a selective inhibitor of transcription, abolished the incorporation of 2-[(14)C]uridine into cellular RNA and significantly reduced the incorporation of L-U-[(14)C]phenylalanine into cellular protein under control conditions and in response to ET-1. The selective antagonists at the ET(A) receptor [BQ123 (100 nM) and PD155080 (100 nM)] and the selective antagonist at the ET(B) receptor [BQ788 (100 nM)] significantly reduced the incorporation of L-U-[(14)C]phenylalanine into cellular protein in response to ET-1 (10 nM). The selective inhibitor of protein kinase C (PKC), bisindolylmaleimide (BIM) (5 microM), reduced markedly the incorporation of 2-[(14)C]uridine into cellular RNA and, to a lesser degree, the incorporation of L-U-[(14)C]phenylalanine into cellular protein in response to ET-1 (100 pM to 10 nM). ET-1 exerts hypertrophic effects directly in vitro in ventricular cardiomyocytes isolated from the hearts of adult rabbits. These effects are (a) due to de novo synthesis since total mass of cellular protein and incorporation of L-U-[(14)C]phenylalanine and 2-[(14)C]uridine into cellular protein and RNA, respectively, were increased; (b) mediated by both the ET(A) and ET(B) receptor subtypes; and (c) may be associated, at least partly, with the activation of PKC.

Use of a-192621 to provide evidence for involvement of endothelin ETB-receptors in endothelin-1-mediated cardiomyocyte hypertrophy

Increased plasma levels of endothelin-1 correlate with the severity of left ventricular hypertrophy in vivo. The aim of the study was to determine the relative contribution of stimulation of endothelin ET(A) and endothelin ET(B) receptors, and the associated activation of protein kinase C, to the hypertrophic response initiated by endothelin-1 in adult rat ventricular cardiomyocytes maintained in culture (24 h). Endothelin-1 (10(-7) M) increased the total mass of protein and the incorporation of [14C] phenylalanine into protein to 26% and 25% greater (P<0.05) than respective basal values. The total content of RNA and the incorporation of 2-[14C] uridine into RNA were increased by 23% and 21%, respectively, by endothelin-1 (10(-8) M). Actinomycin D (5x10(-6) M), an inhibitor of transcription, abolished the incorporation of [14C] phenylalanine and the increased protein mass elicited by endothelin-1 (10(-8) M). The selective agonists at the endothelin ET(B) receptor, sarafotoxin 6c (10(-7) M) and endothelin-3 (10(-7) M), increased the incorporation of [14C] phenylalanine to 13% and 13% greater than respective basal values. The incorporation of [14C]phenylalanine in response to endothelin-1 (10(-7) M) was reduced by 50% (P<0.05) by the selective antagonist at endothelin ET(A) receptors, ABT-627 (10(-9) M), while the response to sarafotoxin 6c was not attenuated. The selective antagonist at endothelin ET(B) receptors, A192621 (10(-10) M), abolished the response to sarafotoxin 6c (10(-7) M) and attenuated the response to endothelin-1 (10(-7) M) by 43% (P<0.05). The selective inhibitor of protein kinase C, bisindolylmaleimide (5x10(-6) M) attenuated the response to sarafotoxin 6c (10(-7) M) by 78% and that to endothelin-1 (10(-7) M), elicited in the presence of A192621 (10(-10) M), by 52%. In conclusion, these data implicate endothelin ET(B) receptors, in addition to endothelin ET(A) receptors, in endothelin-1-mediated cardiomyocyte hypertrophy and provide evidence for the involvement of protein kinase C, at least in part, in the hypertrophic signalling pathways associated with activation of each receptor subpopulation.

Actions of the novel vasodilator, flosequinan, in isolated ventricular cardiomyocytes

Summary Although the potent vasodilating effect of flosequinan is well characterised, the positive inotropic action reported is more varied and less well understood. We examined the contractile and electrophysiologic effects of flosequinan and its metabolite. BTS 53554, in cardiomyocytes from either adult male Sprague-Dawley rats (200–250 g) or New-Zealand White rabbits (2–2.5 kg) and compared the effects with those of sulmazole and enoximone [selective phosphodiesterase (PDE) III inhibitors], Ro 20–1724 and rolipram (selective PDE IV inhibitors) and 3-isobutyl-l-methylxanthine (IBMX, nonselective PDE inhibitor). Flosequinan and BTS 53554 had positive contractile effects (p < 0.05) in both rat and rabbit ventricular cardiomyocytes only at the maximum concentration (10-3 M). Differences were noted between species, however. Flosequinan 10-3 M had a greater contractile effect than BTS 53554 (10-3 M) in rabbit cardiomyocytes, but not in rat cardiomyocytes. We studied the interaction of flosequinan or the metabolite with other PDE inhibitors in rat cardiomyocytes. Contractile amplitudes were not significantly different with equimolar concentrations (3 × 10 4 M) of Ro 20–1724, flosequinan, or BTS 53554 alone (15 ± 6. 18 ± 4. and 32 ± 10%, respectively, greater than the mean basal dL value of 7.38 ± 0.12%, mean ± SE error). However, the combinations of Ro 20–1724 with flosequinan and Ro 20–1724 with BTS 53554 produced synergistic responses: 71 ± 10 and 72 ± 14%, respectively, greater than the mean basal dL value (p < 0.05). In contrast, the combinations of either flosequinan or BTS 53554 with IBMX or sulmazole produced no further increase in contractile amplitude. Neither flosequinan nor BTS 53554 produced any detectable increase in cyclic AMP, whereas significant increases were noted with Ro 20–1724, IBMX, and sulmazole (p < 0.05) in rat cardiomyocytes. Flosequinan increased beating frequency in rat isolated right auricles concentration dependently and was significant over the concentration range of 10-5-3 × 10-4 M; flosequinan 3 × 10 4 M maximally increased the mean frequency of beating by 35% of the predrug value (255 ± 15 beats/min). Flosequinan had no effect on resting membrane potential, amplitude, or maximum upstroke velocity in rat isolated left ventricular (LV) papillary muscle, but at the maximum concentration (10 3 M), flosequinan decreased action potential duration (APD) at 10, 50, and 75% of repolarization (p < 0.05). BTS 53554 produced no changes in AP characteristics over the concentration range of 10 5-10-3 M. The positive contractile and electrophysiologic effects of flosequinan and its major metabolite. BTS 53554, result from selective inhibition of the PDE III isoenzyme. However, these effects were observed only at increased concentrations and probably have no clinical value. Flosequinan had a more pronounced effect on chronotropy (10-5-3 × 10 4 M) than on contractile function, which was influenced significantly only at an increased concentration (10-3 M). Potentiation of the response to rolipram, in terms of APD prolongation, may be caused by a novel mechanism of action such as enhanced intracellular release of Ca2+ by flosequinan.

Cardiotonic actions of selective phosphodiesterase inhibitors in rat isolated ventricular cardiomyocytes.

1 The contractile effects of the novel cardiotonic agent HN‐10200 (2‐[3‐methoxy‐5‐methylsulphinyl‐2‐thienyl]‐1H‐imidazo‐[4,5‐c]‐pyridine hydrochloride), were examined and comparisons made with the responses obtained to a structurally similar compound, sulmazole, and to a number of other compounds which are known to inhibit phosphodiesterase (PDE) isoenzymes with differing selectivities; namely, enoximone (PDE III inhibitor), Ro 20–1724 (PDE IV inhibitor) and 3‐isobutyl‐1‐methylxanthine (non‐selective PDE inhibitor). 2 Contractile function, as measured by mechanical shortening, and biochemical systems involving cyclic AMP were investigated in ventricular cardiomyocytes isolated from adult Sprague‐Dawley rats (200–250 g). 3 HN‐10200 exerted a concentration‐dependent (10−8 m − 10−4 m) positive contractile effect, which was independent of α‐ or β‐adrenoceptor, or histamine receptor stimulation. 4 The efficacies of the contractile responses to the PDE inhibitors were of the order: HN‐10200 > IBMX > sulmazole > enoximone and maximum stimulations, which were obtained at concentrations of 10−4 m, were 54 ± 4%, 41 ± 7%, 38 ± 7% and 26 ± 5% (mean ± s.e.) greater than basal levels, respectively (n = 6); the basal value of contractile amplitude (dL), in the absence of PDE inhibitors was 7.39 ± 0.18% (mean ± s.e.). Ro 20–1724 did not have any effect on contractile activity. 5 Due to low basal levels of cyclic nucleotides in isolated cells, accumulation of cyclic AMP due to the presence of the PDE inhibitors was detected only when the levels of cyclic nucleotide were enhanced with forskolin (10 μm). 6 The PDE inhibitors increased levels of cyclic AMP only at concentrations > 10−4 m. HN‐10200 and sulmazole had similar concentration‐dependent profiles for the accumulation of cyclic AMP; their potencies were lower than that of IBMX (concentrations of forskolin required to increase cyclic AMP by 4 pmol mg−1 protein, in the presence of maximum concentrations of the PDE inhibitors, were 13 ± 3 μm, 14 ± 3 μm and 3 ± 0.6 μm [mean ± s.e.], respectively). 7 These results indicate that a similar mechanism, probably through a weak inhibition of the cyclic AMP‐specific PDE isoenzymes, is responsible for the increase in levels of cyclic AMP by HN‐10200 and sulmazole. However, cyclic AMP is only partially responsible for the positive contractile effect of HN‐10200 and, similarly, sulmazole and IBMX. The lack of apparent increase in levels of cyclic AMP by enoximone, highlights its degree of selectivity for the PDE III isoenzyme, such that the PDE IV isoform is still present in sufficient quantity to degrade cyclic AMP within the cell. On the other hand, the potent action of Ro 20–1724 on accumulation of cyclic AMP, in addition to the lack of effect on contractile function, is in agreement with the selectivity of this compound for the PDE IV isoenzyme and compartmentalization of cyclic AMP in rat isolated ventricular cardiomyocytes.

SRIF Receptor Subtype Expression and Involvement in Positive and Negative Contractile Effects of Somatostatin-14 (SRIF-14) in Ventricular Cardiomyocytes

Background/Aims: Somatostatin-14 (SRIF-14), a neuropeptide co-stored with acetylcholine in the cardiac parasympathetic innervation, exerts both positive and negative influences directly on contraction of ventricular cardiomyocytes, indicative of involvement of more than one of five known SRIF (SSTR) receptor subtypes. The aim was to characterize receptor subtype expression in adult rat ventricular cardiomyocytes and to investigate the influence of a series of SRIF (SSTR) subtype-selective agonists on contractile parameters. Methods: mRNA and protein expression of each receptor subtype were quantified by RT-PCR and immunoblotting respectively; for contraction studies, cells were stimulated at 0.5 Hz under basal conditions and in the presence of isoprenaline (ISO, 10-8M). Results: all five SRIF (SSTR) receptor subtypes were expressed in cardiomyocytes although SRIF1A (SSTR2) and SRIF2A (SSTR1) were less abundant than the other subtypes. L803087 (10-8M), a SRIF2B (SSTR4) agonist, attenuated ISO-stimulated peak contractile amplitude and prolonged relaxation time (T50). L796778 (10-7M), a SRIF1C (SSTR3) agonist, augmented basal and ISO-stimulated peak contractile amplitude; L779976 (10-8M) and L817818 (10-9M), agonists at SRIF1A (SSTR2) and SRIF1B (SSTR5) receptors, respectively, also augmented ISO-stimulated peak amplitude. Conclusion: these data support involvement of SRIF2B (SSTR4) receptors in the negative contractile effects of SRIF-14, while one or more of the three SRIF1 receptor subtypes (SSTR2, 3 or 5) may contribute to the positive contractile effects of SRIF-14.

Modulation of contractile function through neuropeptide Y receptors during development of cardiomyocyte hypertrophy

Severity of left ventricular hypertrophy (LVH) correlates with elevated plasma levels of neuropeptide Y (NPY) in hypertension. NPY elicits positive and negative contractile effects in cardiomyocytes through Y1 and Y2 receptors, respectively. This study tested the hypothesis that NPY receptor-mediated contraction is altered during progression of LVH. Ventricular cardiomyocytes were isolated from spontaneously hypertensive rats (SHRs) pre-LVH (12 weeks), during development (16 weeks), and at established LVH (20 weeks) and age-matched normotensive Wistar Kyoto (WKY) rats. Electrically stimulated (60 V, 0.5 Hz) cell shortening was measured using edge detection and receptor expression determined at mRNA and protein level. The NPY and Y1 receptor-selective agonist, Leu31Pro34NPY, stimulated increases in contractile amplitude, which were abolished by the Y1 receptor-selective antagonist, BIBP3226 [R-N2-(diphenyl-acetyl)-N-(4-hydroxyphenyl)methyl-argininamide)], confirming Y1 receptor involvement. Potencies of both agonists were enhanced in SHR cardiomyocytes at 20 weeks (2300- and 380-fold versus controls). Maximal responses were not attenuated. BIBP3226 unmasked a negative contraction effect of NPY, elicited over the concentration range (10–12 to 3 × 10–9 M) in which NPY and PYY3–36 attenuated the positive contraction effects of isoproterenol, the potencies of which were increased in cardiomyocytes from SHRs at 20 weeks (175- and 145-fold versus controls); maximal responses were not altered. Expression of NPY-Y1 and NPY-Y2 receptor mRNAs was decreased (55 and 69%) in left ventricular cardiomyocytes from 20-week-old SHRs versus age-matched WKY rats; parallel decreases (32 and 80%) were observed at protein level. Enhancement of NPY potency, producing (opposing) contractile effects on cardiomyocytes together with unchanged maximal response despite reduced receptor number, enables NPY to contribute to regulating cardiac performance during compensatory LVH.

Mechanical effects of ET-1 in cardiomyocytes isolated from normal and heart-failed rabbits.

Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca2+ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca2+ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n=7) or with saline (n=7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca2+ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 ± 11% (mean ± S.D.) in control cardiomyocytes and 33 ± 6% in heart-failed cells. However, there was no significant change in the EC50 obtained with ET-1 for healthy (0.31 ± 0.1 nM) and for failed cardiomyocytes (0.24 ± 0.1 nM). The effects of ET-1 on L-type Ca2+ channels were similar with a peak amplitude at 1 nM ET-1 of −3.26 ± 0.8 ⋬ in control cardiomyocytes and −3.32 ± 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca2+ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.