Martin N. Hicks

Overexpression of the 5-Hydroxytryptamine Transporter Gene Effect on Pulmonary Hemodynamics and Hypoxia-Induced Pulmonary Hypertension

Background—Increased serotonin (5-hydroxytryptamine, 5-HT) transporter activity has been observed in human familial pulmonary hypertension. Methods and Results—We investigated pulmonary hemodynamics and the development of hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in mice overexpressing the gene for the 5-HT transporter (5-HTT+ mice). Right ventricular pressure was elevated 3-fold in normoxic 5-HTT+ mice compared with their wild-type controls. Hypoxia-induced increases in right ventricular hypertrophy and pulmonary vascular remodeling were also potentiated in the 5-HTT+ mice. 5-HTT–like immunoreactivity, protein, and binding sites were markedly increased in the lungs from the 5-HTT+ mice. Hypoxia, however, decreased 5-HT transporter immunoreactivity, mRNA transcription, protein, and binding sites in both wild-type and 5-HTT+ mice. Conclusions—Increased 5-HT transporter expression causes elevated right ventricular pressures, and this occurs before the onset of right ventricular hypertrophy or pulmonary arterial remodeling. Hypoxia-induced remodeling is, however, increased in 5-HTT+ mice, whereas hypoxia inhibits 5-HTT expression. This provides a unique model that demonstrates differential mechanisms for familial pulmonary arterial hypertension and pulmonary arterial hypertension with hypoxemia.

Functional, structural, and dynamic basis of electrical heterogeneity in healthy and diseased cardiac muscle: implications for arrhythmogenesis and anti-arrhythmic drug therapy☆

The electrophysiological properties of the ventricular myocardium are extremely heterogeneous. There are intrinsic electrical differences between the myocytes from different regions of the heart (most notably between the epicardium, midmyocardium, and endocardium), which are the result of different contributions of ionic currents to the transmembrane action potential. Sources of local anisotropy include directional differences in the distribution of gap junctions between adjacent myocytes and the presence of intercalated non-myocytes (e.g., fibroblasts), propagation boundaries, and wavefront collisions, which can lead to local variability of electrical load and, therefore, to nonuniform depolarisation and repolarisation. In addition, the complex anatomical arrangement of the myocardial fibres and nonuniform distribution of transmural mechanical stresses also contribute to electrical heterogeneity. Finally, dispersion of repolarisation is dynamically modified by the restitution properties of individual myocytes, stimulation rate, and the direction of conduction. All aspects of this electrical heterogeneity can be affected by different pathological conditions, such as myocardial ischaemia and cardiac hypertrophy. In particular, differential responses of various myocyte populations to these pathological stimuli and a marked increase in nonuniform anisotropy may be responsible for increased pro-arrhythmic potential in these conditions. In addition, the clinical effectiveness of anti-arrhythmic drugs may be related to their effects on electrical heterogeneity.

Control of Cardiac-specific Transcription by p300 through Myocyte Enhancer Factor-2D

The transcriptional integrator p300 regulates gene expression by interaction with sequence-specific DNA-binding proteins and local remodeling of chromatin. p300 is required for cardiac-specific gene transcription, but the molecular basis of this requirement is unknown. Here we report that the MADS (MCM-1, agamous, deficiens, serum response factor) box transcription factor myocyte enhancer factor-2D (MEF-2D) acts as the principal conduit for cardiac transcriptional activation by p300. p300 activation of the native 2130-base pair human skeletal α-actin promoter required a single hybrid MEF-2/GATA-4 DNA motif centered at −1256 base pairs. Maximal expression of the promoter in cultured myocytes and in vivo correlated with binding of both MEF-2 and p300, but not GATA-4, to this AT-rich motif. p300 and MEF-2 were coprecipitated from cardiac nuclear extracts by an oligomer containing this element. p300 was found exclusively in a complex with MEF-2D at this and related sites in other cardiac-restricted promoters. MEF-2D, but not other MEFs, significantly potentiated cardiac-specific transcription by p300. No physical or functional interaction was observed between p300 and other factors implicated in skeletal actin transcription, including GATA-4, TEF-1, or SRF. These results show that, in the intact cell, p300 interactions with its protein targets are highly selective and that MEF-2D is the preferred channel for p300-mediated transcriptional control in the heart.

Regulated expression of a foreign gene targeted to the ischaemic myocardium

OBJECTIVES Regulated expression of transferred foreign genes may be an important feature of gene therapy. Because coronary artery disease often involves intermittent myocardial ischaemia followed by periods of normal cardiac function it will probably be necessary to regulate the expression of putative therapeutic/cardioprotective genes directly in response to ischaemia-associated signals. The objectives of the current study were to develop a combination of gene regulatory components that can be used to target a product to the myocardium and limit the expression of the gene to periods of ischaemic activity. METHODS Expression plasmids were constructed containing muscle-specific promoters and hypoxia-responsive enhancer elements linked to a reporter gene. The regulation of these constructs by hypoxia or experimental ischaemia was measured following transient expression in cultured cells or after direct injection of DNA into the rabbit myocardium. RESULTS A single set of hypoxia response elements placed immediately upstream of the minimal muscle-specific alpha-myosin heavy chain promoter conferred potent positive regulation of this promoter by hypoxia in vitro and by ischaemia in vivo. Induction by ischaemia persisted for at least 4 h and returned to the baseline level within 8 h. CONCLUSIONS Hypoxia responsive regulatory elements, in combination with weak tissue-restricted promoters incorporated into an appropriate vector system may allow controlled expression of a therapeutic gene in ischaemic myocardium.

Pulmonary hypertension secondary to left ventricular dysfunction: the role of nitric oxide and endothelin-1 in the control of pulmonary vascular tone

Using an in vivo model of pulmonary hypertension (PHT) secondary to left ventricular dysfunction (LVD), the pulmonary arterial response to the nitric oxide synthase (NOS) blocker L‐NAME (30 μmol.min−1 i.v.) and the subsequent responses to cumulatively administered endothelin‐1 (ET‐1) (0.001 – 4 nmol.kg−1 i.v.) or big ET‐1 (0.1 – 2.0 nmol.kg−1 i.v.) were studied. Additionally, the effect of the non‐selective ET‐1 receptor antagonist, SB209670, was investigated. Eight weeks after coronary artery ligation or sham operation, rabbits demonstrated increased mean pulmonary arterial pressure (PAP) accompanied by right ventricular hypertrophy. Blockade of NOS caused a greater increase in basal PAP (increased by 7.7±1.1 mmHg c.f. 3.8±1.0 mmHg in controls, P<0.05) and uncovered a greater pulmonary pressor response to exogenous ET‐1 in rabbits with PHT (increased by 10.2±2.3 mmHg c.f. 4.9±1.0 mmHg in controls, P<0.05). Big ET‐1 evoked a pulmonary pressor effect, in both groups of rabbits, that was increased following blockade of NOS and was more potent in rabbits with PHT. The non‐selective ET‐1 receptor antagonist, SB209670, reduced basal PAP (from 16.9 mmHg to 15.9 mmHg, P<0.05) in rabbits with PHT and blocked the response to ET‐1 in the presence of L‐NAME. In conclusion, the results demonstrate that basal NO activity masks a pulmonary pressor response to exogenously administered ET‐1. An increased responsiveness to ET‐1 was shown in the pulmonary arterial bed of rabbits with PHT secondary to LVD, implicating a pathophysiological role for ET‐1 in this model.

Regional electrophysiological effects of left ventricular hypertrophy in isolated rabbit hearts under normal and ischaemic conditions

OBJECTIVES Left ventricular hypertrophy (LVH) has been reported to produce differential electrophysiological effects in isolated epicardial and endocardial cells. This study aimed to examine regional electrophysiological effects of LVH in normal and ischaemic conditions in the whole heart. METHODS LVH was secondary to perinephritis-induced hypertension. Monophasic action potential duration (MAPD(90)), effective refractory period (ERP) and conduction delay were measured in paced, isolated working rabbit hearts either at one right ventricular and two left ventricular sites (apical and basal epicardium) or at three left ventricular sites (apical and basal epicardium, apical endocardium). The hearts were subjected to 30 min of regional ischaemia and 15 min of reperfusion. RESULTS In non-ischaemic conditions, LVH produced uniform prolongation of MAPD(90) and ERP in the left ventricular epicardium, but not in the endocardium. After coronary artery occlusion, LVH significantly increased ischaemia-induced transepicardial dispersion of repolarisation, but not refractoriness. LVH did not affect arrhythmogenesis in either non-ischaemic or ischaemic conditions. CONCLUSIONS Differential effects of LVH on epicardial and endocardial electrophysiological parameters are also observed in the whole heart. In addition, the sensitivity of hypertrophied myocardium to ischaemia is increased and leads to an increase in ischaemia-induced dispersion of repolarisation. However, neither dispersion of refractoriness nor arrhythmogenesis are affected by LVH in non-ischaemic or ischaemic conditions in this experimental model.

Regional electrophysiological effects of hypokalaemia, hypomagnesaemia and hyponatraemia in isolated rabbit hearts in normal and ischaemic conditions

OBJECTIVE The aims of this study were to establish an isolated working heart model for electrophysiological recordings from the epicardium and endocardium and to examine regional effects of changes in ion concentrations in normal and ischaemic conditions. METHODS Monophasic action potential duration (MAPD90), effective refractory period (ERP) and conduction delay were measured simultaneously in the epicardium and endocardium of rabbit hearts paced at 3.3 Hz, subjected to 30 min of regional ischaemia and 15 min of reperfusion. The hearts were exposed before and throughout ischaemia and reperfusion to hypokalaemia (K+ = 2 mM), hypomagnesaemia (Mg2+ = 0.5 mM) or hyponatraemia (Na+ = 110 mM). RESULTS In the control hearts, no regional electrophysiological differences were seen before ischaemia, but ischaemia-induced MAPD90 shortening and postrepolarisation refractoriness were greater in the epicardium than in the endocardium and conduction delay increased only in the epicardium. Hypokalaemia shortened ERP in the epicardium (but not endocardium) and increased conduction delay in all areas before ischaemia, but it had no effects during ischaemia. During reperfusion hypokalaemia increased the incidence of recurrent tachyarrhythmias. Hypomagnesaemia had no effect before ischaemia, increased epicardial (but not endocardial) MAPD90 shortening during ischaemia, although it had no pro-arrhythmic action. Hyponatraemia increased conduction delay in all areas before ischaemia and produced asystole or severe bradycardia in all hearts. During ischaemia, hyponatraemia decreased ERP shortening and inducibility of arrhythmias in the epicardium (but not endocardium). CONCLUSIONS We conclude that the more pronounced effect of ischaemia upon the epicardium than the endocardium can be explained by the contact of the endocardium with intracavitary perfusate. We also conclude that changes in ion concentrations may have differential regional electrical effects in normal or ischaemic conditions.

Relevance of inter- and intraventricular electrical dispersion to arrhythmogenesis in normal and ischaemic rabbit myocardium: a study with cromakalim, 5-hydroxydecanoate and glibenclamide.

This study aimed to investigate the role of electrical dispersion in arrhythmogenesis by using K(ATP) channel modulating agents. Monophasic action-potential duration (MAPD90), effective refractory period (ERP), and conduction delay were measured at three ventricular sites in isolated working rabbit hearts. Cromakalim (10 microM), glibenclamide (3 microM), or 5-hydroxydecanoate (100 microM) were administered before and throughout 30 min of regional ischaemia and 15 min of reperfusion. Before ischaemia, cromakalim reduced MAPD90 and ERP in all areas and facilitated induction of ventricular fibrillation in five of 12 hearts. In these hearts, cromakalim increased interventricular ERP dispersion from 17 +/- 5 to 38 +/- 5 ms. During ischaemia, cromakalim decreased MAPD90 dispersion within the left ventricle from 84 +/- 5 to 44 +/- 4 ms, but did not affect ERP dispersion and arrhythmogenesis. 5-Hydroxydecanoate had no effect on MAPD90 and ERP shortening or dispersion during ischaemia and reperfusion and was not antiarrhythmic. Glibenclamide reduced forward flow to zero, preventing further electrophysiologic studies. In conclusion, in this model, an increase in interventricular ERP dispersion predisposes to ventricular fibrillation in normoxic conditions after cromakalim administration. However, a decrease in ischaemia-induced MAPD90 dispersion by cromakalim does not affect arrhythmogenesis. A lack of effect of 5-hydroxydecanoate on electrical dispersion during ischaemia is accompanied by a lack of antiarrhythmic activity.

Pulmonary responses to 5-hydroxytryptamine and endothelin-1 in a rabbit model of left ventricular dysfunction

OBJECTIVE To determine whether pulmonary hypertension developed in a coronary artery-ligated rabbit model of left ventricular dysfunction (LVD) and to examine the effects of i.v. 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1) on pulmonary arterial pressure (PAP). METHODS Eight weeks after experimental coronary artery ligation or sham operation, ejection fractions were assessed by echocardiography. The rabbits were later anaesthetised and pulmonary arterial pressure was measured via a catheter inserted into the pulmonary artery via the right external jugular vein. 5-HT (1-400 micrograms/kg) and ET-1 (0.001-4 nmol/kg) were administered i.v. RESULTS Ejection fraction was significantly decreased from 76.6 +/- 1.4% in sham-operated to 42.2 +/- 1.3% in coronary artery-ligated rabbits (n = 9 in each group; P < 0.001), consistent with LVD. Baseline mean pulmonary arterial pressure was significantly increased in the coronary artery-ligated group compared to the shams, (16.5 +/- 0.5 vs. 11.5 +/- 0.8 mmHg; P < 0.001). A significant degree of right ventricular hypertrophy was found in the coronary artery-ligated rabbits (0.70 +/- 0.04 g/kg final body weight (f.b.wt.), n = 8 cf. 0.48 +/- 0.02 g/kg f.b.wt. in sham-operated controls, n = 8; P < 0.001). There was a significant increase in the percentage of muscularised pulmonary vessels adjacent to alveolar ducts and alveoli < 60 microns i.d. in the LVD rabbits compared with their sham-operated controls (8.5 +/- 0.4 cf. 20 +/- 0.5%; P < 0.0005). 5-HT produced a greater response in the coronary artery-ligated rabbits (a maximum increase of 8.7 +/- 1.0 mmHg in mean pulmonary artery pressure vs. 4.6 +/- 1.5 mmHg for sham-operated controls; P < 0.05). ET-1 did not have any effect on pulmonary arterial pressure in either group. CONCLUSION In the rabbit, LVD secondary to coronary artery ligation, causes right ventricular hypertrophy, pulmonary vascular remodelling, and an increased PAP consistent with the onset of pulmonary hypertension (PHT). The greater PAP response to i.v. 5-HT in the PHT group supports the hypothesis that this substance could be involved in the development of PHT. A role for ET-1 cannot be excluded, despite its lack of effect on PAP when intravenously administered in either group.

The electrophysiology of rabbit hearts with left ventricular hypertrophy under normal and ischaemic conditions

OBJECTIVES To examine the cardiac electrophysiological effects of left ventricular hypertrophy (LVH) and to determine whether any observed differences are modified by global zero-flow ischaemia. METHODS LVH was induced by perinephritic hypertension in New Zealand White rabbits. Transmembrane action potential recordings were made using conventional floating glass microelectrodes and effective refractory periods (ERP) determined by programmed stimulation in isolated arterially perfused interventricular septa during normal perfusion and a 30-min period of global ischaemia. The electrophysiological data were pooled into 6-min periods during ischaemia. RESULTS The post-operative blood pressure was 76(2) mmHg (mean(s.e.m.)) and 113(2) mmHg (P < 0.0005) in the sham and perinephritic rabbits respectively. The left ventricular to body weight ratio was 0.27(0.01) g kg-1 in the sham and 0.36(0.02) g kg-1 in the perinephritic group (P < 0.005) representing 33% hypertrophy. In the isolated septa, prior to ischaemia, the hypertrophied group exhibited significant prolongations in action potential duration to 50% and 90% repolarisation (APD50, APD90) and ERP of 20%, 12% and 19% respectively (P < 0.005) without any differences in resting membrane potential (Em), upstroke velocity (dV/dtmax) or amplitude (APA) of the action potential. During ischaemia Em, APA and dV/dtmax progressively decreased to a similar extent in both groups. Ischaemia resulted in shortenings in APD50, APD90 and ERP in the hypertrophy group of 122(9) ms, 131(8) ms and 99 (6) ms respectively which were greater than those observed in the control group (84 (7) ms, 115 (7) ms and 50 (13) ms, P < 0.05). These differences resulted in loss of the preischaemic prolongation of repolarisation and refractoriness in the hypertrophy group. CONCLUSIONS There was enhanced shortening of APD and ventricular refractoriness in hypertrophied muscle during global ischaemia. This could increase the dispersion of repolarization and refractoriness between normal and ischaemic hypertrophied muscle during regional ischaemia which may explain the increased susceptibility of hypertrophied hearts to arrhythmias.