George Hart

Exercise training reduces resting heart rate via downregulation of the funny channel HCN4

Endurance athletes exhibit sinus bradycardia, that is a slow resting heart rate, associated with a higher incidence of sinus node (pacemaker) disease and electronic pacemaker implantation. Here we show that training-induced bradycardia is not a consequence of changes in the activity of the autonomic nervous system but is caused by intrinsic electrophysiological changes in the sinus node. We demonstrate that training-induced bradycardia persists after blockade of the autonomous nervous system in vivo in mice and in vitro in the denervated sinus node. We also show that a widespread remodelling of pacemaker ion channels, notably a downregulation of HCN4 and the corresponding ionic current, If. Block of If abolishes the difference in heart rate between trained and sedentary animals in vivo and in vitro. We further observe training-induced downregulation of Tbx3 and upregulation of NRSF and miR-1 (transcriptional regulators) that explains the downregulation of HCN4. Our findings provide a molecular explanation for the potentially pathological heart rate adaptation to exercise training.

Contrast enhanced micro-computed tomography resolves the 3-dimensional morphology of the cardiac conduction system in mammalian hearts.

The general anatomy of the cardiac conduction system (CCS) has been known for 100 years, but its complex and irregular three-dimensional (3D) geometry is not so well understood. This is largely because the conducting tissue is not distinct from the surrounding tissue by dissection. The best descriptions of its anatomy come from studies based on serial sectioning of samples taken from the appropriate areas of the heart. Low X-ray attenuation has formerly ruled out micro-computed tomography (micro-CT) as a modality to resolve internal structures of soft tissue, but incorporation of iodine, which has a high molecular weight, into those tissues enhances the differential attenuation of X-rays and allows visualisation of fine detail in embryos and skeletal muscle. Here, with the use of a iodine based contrast agent (I2KI), we present contrast enhanced micro-CT images of cardiac tissue from rat and rabbit in which the three major subdivisions of the CCS can be differentiated from the surrounding contractile myocardium and visualised in 3D. Structures identified include the sinoatrial node (SAN) and the atrioventricular conduction axis: the penetrating bundle, His bundle, the bundle branches and the Purkinje network. Although the current findings are consistent with existing anatomical representations, the representations shown here offer superior resolution and are the first 3D representations of the CCS within a single intact mammalian heart.

Normal regional distribution of membrane current density in rat left ventricle is altered in catecholamine-induced hypertrophy

OBJECTIVE To test the hypothesis that changes in the normal regional distribution of potassium and calcium currents contribute to the different regional changes in action potential duration in isoprenaline-induced hypertrophy in rats. METHODS Hypertrophy was elicited in rats by seven daily injections of isoprenaline. Left ventricular myocytes were isolated from basal sub-endocardial, basal mid-myocardial and apical sub-epicardial tissue. Membrane currents were measured using the whole-cell patch-clamp technique at 35 +/- 1 degrees C. RESULTS Cell membrane capacitance was similar in all three groups and was increased by 17% in hypertrophy (P < 0.001, t-test). Changes in the calcium-independent transient outward current (Ito1) density in hypertrophy were different in the three regions (P < 0.05, ANOVA). Ito1 was reduced in sub-epicardial (control, 23.4 +/- 2.0 pA pF-1; hypertrophy, 15.8 +/- 1.5 pA pF-1, P < 0.01 ANOVA) and in mid-myocardial myocytes (control, 24.0 +/- 2.8 pA pF-1; hypertrophy, 13.8 +/- 1.3 pA pF-1, P < 0.01 ANOVA) and was not significantly altered in sub-endocardial myocytes (control, 8.5 +/- 0.7 pA pF-1; hypertrophy, 7.4 +/- 1.8 pA pF-1). Steady-state background current density was reduced in hypertrophy (P < 0.05, ANOVA). The regional difference in steady-state background current in control hearts (P < 0.05, ANOVA) was altered in hypertrophy. Calcium current (ICa) density was similar in the three regions studied in both control and hypertrophied hearts. ICa was reduced in hypertrophy (P < 0.05, ANOVA). CONCLUSION The normal regional differences in Ito1 are reduced, in steady-state background current are altered and in ICa are unchanged in catecholamine-induced hypertrophy in the rat left ventricle. These data may in part explain the reduction in the normal regional differences in APD observed in hypertrophy.

Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands

SUMMARY Horse flies feed from superficial haematomas and probably rely heavily on the pharmacological properties of their saliva to find blood. Here we describe the first evidence of vasodilators in horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary gland extract and clone and express one of the active peptides (termed vasotab). Physiological tests using crude salivary gland extracts and reverse-phase HPLC fractions demonstrated positive inotropism in isolated rat hearts, vasodilatation of coronary and peripheral vessels, and Na, K-ATPase inhibition. One of the vasoactive fractions was analysed by N-terminal Edman degradation and a 47-amino-acid sequence obtained. A full-length cDNA encoding the peptide was cloned from a phage library using degenerate primer PCR and the peptide expressed in insect cells. A 20-amino-acid signal sequence precedes the mature 56-amino-acid vasotab peptide, which is a member of the Kazal-type protease inhibitor family. The peptide has a unique 7-amino-acid insertion between the third and fourth cysteine residues. The recombinant peptide prolonged the action potential and caused positive inotropism of isolated rat heart myocytes, and may be an ion channel modulator.

Greater antiarrhythmic activity of acute 17β-estradiol in female than male anaesthetized rats: correlation with Ca2+ channel blockade

Female sex hormones may protect pre‐menopausal women from sudden cardiac death. We therefore investigated the effects of the main female sex hormone, 17β‐estradiol, on ischaemia‐induced cardiac arrhythmias and on the L‐type Ca2+ current (ICaL).

A topographical study of mechanical and electrical properties of single myocytes isolated from normal guinea-pig ventricular muscle

Major regional differences in the electrical properties of myocytes from ventricular muscle have been described previously, on the basis of samples taken from a maximum of three regions in each heart. In order to define the topographical basis for such differences, we studied the electrical and mechanical properties of single myocytes isolated from 20 regions throughout the ventricles in the normal guinea‐pig heart. Single myocytes were isolated using an enzymatic dispersion method, and were studied under conditions that were close to physiological. Cell capacitance and action potentials were recorded using the switch‐clamp technique, and cell length and evoked shortening were measured using a photodiode array system. In the left ventricular free wall, mid‐myocardial cells were longer and had greater capacitative surface area than surface myocytes. There were transmural but not longitudinal differences in APD90 (action potential duration to 90% repolarization), with the longest APD90 in subendocardial and the shortest in subepicardial myocytes. We found a septum – left ventricular free wall – right ventricular free wall gradient, with the longest APD90 in the septum and the shortest in the right ventricular free wall. The regional distribution of APD90 was closely mirrored by relaxation time. Peak cell shortening was greater in subendocardial myocytes than in subepicardial myocytes in the left ventricular free wall, and in myocytes from the left side of the septum compared with the right. We concluded that the regional distribution of APD is closely and inversely related to the sequence of ventricular depolarization, and that the regional variations in cell shortening amplitude are related principally to reported regional variations in wall stress.

A prospective evaluation of cardiac function in patients with chronic myeloid leukaemia treated with imatinib

In vitro studies have suggested that imatinib may be toxic to cardiac myocytes. Though retrospective studies have not shown clinical heart failure, these did not look for subtle cardiac damage. We have carried out a prospective cardiac assessment in 59 chronic myeloid leukaemia (CML) patients treated with imatinib for a median of 3.4 years, using echocardiography and MUGA scanning, with the latter repeated after a further year. We report no evidence of myocardial deterioration, either at baseline or over 12 months of imatinib treatment. Imatinib cardiotoxicity is not an important clinical consideration for CML patients or their advisors.

Molecular and functional distributions of chloride conductances in rabbit ventricle

The regulation of cardiac electrical activity is critically dependent on the distribution of ion channels in the heart. For most ion channels, however, the patterns of distribution and what regulates these patterns are not well characterized. The most likely candidates for the genes that encode the cAMP- and swelling-activated chloride conductances in the heart are an alternatively spliced variant of CFTR and ClC-3, respectively. In this study we have 1) measured the density of CFTR and ClC-3 mRNA levels across the left ventricular free wall (LVFW) of the rabbit heart using in situ hybridization and 2) measured the corresponding current density of cAMP- and swelling-activated chloride channels in myocytes isolated from subepicardial, midmyocardial, and subendocardial regions of the LVFW. There was a highly significant gradient in the whole cell slope conductance of cAMP-activated chloride currents; normalized slope conductance at 0 mV was 15.7 ± 1.8 pS/pF ( n = 9) in subepicardial myocytes, 7.8 ± 1.5 pS/pF ( n = 11) in midmyocardial myocytes, and 4.9 ± 1.1 pS/pF ( n = 9) in subendocardial myocytes. The level of CFTR mRNA was closely correlated with the density of cAMP-activated chloride conductances in different regions of the heart, with the level of CFTR mRNA being three times higher in the subepicardium than in the subendocardium. The whole cell slope conductance of swelling-activated chloride channel activity, measured 3-5 min after the commencement of cell swelling, was higher in myocytes isolated from the subepicardium than in myocytes isolated from the midmyocardium or subendocardium. In contrast, there was a uniform expression of ClC-3 mRNA across the LVFW of the rabbit heart. These results suggest that the control of gene expression is an important contributor in regulating the distribution of cAMP-activated chloride channels in the rabbit heart but that it may be less important for the swelling-activated chloride channels.

The role of constitutive PKA-mediated phosphorylation in the regulation of basal ICa in isolated rat cardiac myocytes

1 Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L‐type Ca2+ current (ICa) observed in the absence of exogenous β‐adrenergic receptor stimulation is sustained by PKA‐mediated phosphorylation. Amphotericin B was used to record whole‐cell ICa in the perforated patch‐clamp configuration. 2 Calyculin A and isoprenaline (both 1 μmol l−1) increased basal ICa (P<0.05), whereas H‐89 inhibited ICa in a concentration‐dependent manner with an IC50 ∼5 μmol l−1. H‐89 also inhibited the response to 1.0 μmol l−1 isoprenaline, although relatively high concentrations (30 μmol l−1) were required to achieve complete suppression of the response. 3 Double‐pulse protocols were used to study the effects of 10 μmol l−1 H‐89 on time‐dependent recovery of ICa from voltage‐dependent inactivation as well as the steady‐state gating of ICa. T0.5 (time for ICa to recover to 50% of the preinactivation amplitude) increased in the presence of H‐89 (P<0.05) but was unaffected by calyculin A or isoprenaline. 4 Steady‐state activation/inactivation properties of ICa were unaffected by 10 μmol l−1 H‐89 or 1 μmol l−1 calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V0.5 for activation and inactivation became more negative. 5 Data show that basal ICa is regulated by cAMP‐PKA‐mediated phosphorylation in the absence of externally applied β‐receptor agonists and that relatively high concentrations of H‐89 are required to fully suppress the response to β‐adrenergic receptor stimulation, thereby limiting the value of H‐89 as a useful tool in dissecting signalling pathways in intact myocytes.

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (INa) and calcium currents (ICa) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that ICa density was unchanged whereas INa density after stepping from −80 to −30 mV was decreased by 30% (−9.0 ± 1.16 pA pF−1 in control and −6.31 ± 0.67 pA pF−1 in hypertrophy, p < 0.05, n= 6). Steady-state activation/inactivation variables of INa, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of INa was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca2+ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in INa density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na+ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased INa density so that net Na+ influx remains unaltered. Changes in the fast INa do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle. (Mol Cell Biochem 261: 217–226, 2004)