Joanne Layland

Role of cyclic GMP‐dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes

Nitric oxide (NO) can directly modulate cardiac contractility by accelerating relaxation and reducing diastolic tone. The intracellular mechanisms underlying these contractile effects are poorly understood. Here we investigate the role of cyclic GMP‐dependent protein kinase (PKG) in the contractile response to exogenous NO in rat ventricular myocytes. Isolated ventricular myocytes were stimulated electrically and contractility was assessed by measuring cell shortening. Some cells were loaded with the fluorescent Ca2+ probe indo‐1 AM for simultaneous assessment of the intracellular Ca2+ transient. The NO donor diethylamine NONOate (DEA/NO, 10 μm) significantly increased resting cell length, reduced twitch amplitude and accelerated time to 50 % relaxation (to 100.8 ± 0.2, 83.7 ± 3.0 and 88.9 ± 3.7 % of control values, respectively). The contractile effects of DEA/NO occurred without significant changes in the amplitude or kinetics of the intracellular Ca2+ transient, suggesting that the myofilament response to Ca2+ was reduced. These effects were abolished by inhibition of either guanylyl cyclase (with 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one; ODQ, 10 μm) or PKG (with Rp‐8‐Br‐cGMPs, 10 μm) suggesting that, at the concentration investigated, the effects of DEA/NO were mediated exclusively by PKG, following activation of guanylyl cyclase and elevation of cGMP. Direct activation of PKG with 8‐pCPT‐cGMP (10 μm) mimicked the effects of DEA/NO (resting cell length and time to 50 % relaxation were 100.6 ± 0.1 and 90.5 ± 1.5 % of control values, respectively).The reduced myofilament Ca2+ responsiveness was not attributable to an intracellular acidosis since the small reduction in pHi induced by DEA/NO was found to be uncoupled from its contractile effects. However, hearts treated with DEA/NO (10 μm) showed a significant increase (1.4‐fold; P < 0.01) in troponin I phosphorylation compared to control, untreated hearts. These results suggest that the reduction in myofilament Ca2+ responsiveness produced by DEA/NO results from phosphorylation of troponin I by PKG.

Positive force- and [Ca2+]i-frequency relationships in rat ventricular trabeculae at physiological frequencies

The isometric force-frequency relationship of isolated rat ventricular trabeculae (diameter <250 μm) was examined at 24, 30, and 37°C at stimulation frequencies (0.1-12 Hz) encompassing the physiological range. Some muscles were microinjected with fura PE3 to monitor the diastolic and systolic intracellular concentration of Ca2+([Ca2+]i). At a near-physiological external Ca2+ concentration ([Ca2+]o) of 1 mM, a positive force-frequency relationship was demonstrated at all temperatures. The force-frequency relationship became negative at high frequencies (e.g., >6 Hz at 30°C) at 1 mM [Ca2+]oor at low frequencies at 8 mM [Ca2+]o. The twitch and Ca2+ transient became shorter as stimulation frequency increased; these changes were related to changes in systolic, rather than diastolic, [Ca2+]iand were not blocked by inhibitors of Ca2+/calmodulin-dependent protein kinase II. The positive force-frequency relationship of rat trabeculae was caused by a frequency-dependent loading of the sarcoplasmic reticulum (SR) with Ca2+. We suggest that at high frequencies, or under conditions of Ca2+ overload, this loading saturates. Processes that tend to decrease SR Ca2+ release will then predominate, resulting in a negative force-frequency relationship.The isometric force-frequency relationship of isolated rat ventricular trabeculae (diameter <250 micrometer) was examined at 24, 30, and 37 degreesC at stimulation frequencies (0.1-12 Hz) encompassing the physiological range. Some muscles were microinjected with fura PE3 to monitor the diastolic and systolic intracellular concentration of Ca2+ ([Ca2+]i). At a near-physiological external Ca2+ concentration ([Ca2+]o) of 1 mM, a positive force-frequency relationship was demonstrated at all temperatures. The force-frequency relationship became negative at high frequencies (e. g., >6 Hz at 30 degreesC) at 1 mM [Ca2+]o or at low frequencies at 8 mM [Ca2+]o. The twitch and Ca2+ transient became shorter as stimulation frequency increased; these changes were related to changes in systolic, rather than diastolic, [Ca2+]i and were not blocked by inhibitors of Ca2+/calmodulin-dependent protein kinase II. The positive force-frequency relationship of rat trabeculae was caused by a frequency-dependent loading of the sarcoplasmic reticulum (SR) with Ca2+. We suggest that at high frequencies, or under conditions of Ca2+ overload, this loading saturates. Processes that tend to decrease SR Ca2+ release will then predominate, resulting in a negative force-frequency relationship.

Essential role of troponin I in the positive inotropic response to isoprenaline in mouse hearts contracting auxotonically

PKA‐dependent phosphorylation of cardiac troponin I (cTnI) contributes significantly to β‐adrenergic agonist‐induced acceleration of myocardial relaxation (lusitropy). However, the role of PKA‐dependent cTnI phosphorylation in the positive inotropic response to β‐adrenergic stimulation is unclear. We studied the contractile response to isoprenaline (10 nm) in isolated hearts and isolated cardiomyocytes from transgenic mice with cardiac‐specific expression of slow skeletal TnI (ssTnI, which lacks the N‐terminal protein extension containing PKA‐sensitive phosphorylation sites in cTnI) and matched wild‐type littermate controls. As expected, the lusitropic effect of isoprenaline was significantly blunted in ssTnI hearts. However, the positive inotropic response to isoprenaline was also blunted in ssTnI hearts. This effect was especially prominent for ejection‐phase indices in isolated auxotonically loaded ssTnI hearts whereas the positive inotropic response of isovolumic hearts or unloaded isolated myocytes was much less affected. Isoprenaline decreased left ventricular end‐systolic volume in wild‐type hearts (10.6 ± 1.6 to 6.2 ± 0.4 μl at a preload of 20 cmH2O; P < 0.05) but not transgenic hearts (11.4 ± 1.3 to 10.9 ± 1.3 μl; P= n.s.). Likewise, isoprenaline increased stroke work in control hearts (14.5 ± 1.0 to 22.5 ± 1.8 mmHg μl mg−1; P < 0.05) but not transgenic hearts (15.4 ± 1.3 to 18.3 ± 1.2 mmHg μl mg−1; P= n.s.). The end‐systolic pressure–volume relation was increased by isoprenaline to a greater extent in control than transgenic hearts. However, isoprenaline induced a similar rise in intracellular Ca2+ transients in transgenic and non‐transgenic cardiomyocytes. These results indicate that cTnI has a pivotal role in the positive inotropic response of the murine heart to β‐adrenergic stimulation, an effect that is highly dependent on loading conditions and is most evident in the auxotonically loaded ejecting heart.

Myofilament-based relaxant effect of isoprenaline revealed during work-loop contractions in rat cardiac trabeculae.

In cardiac muscle, β‐adrenergic stimulation increases contractile force and accelerates relaxation. The relaxant effect is thought to be due primarily to stimulation of Ca2+ uptake into the sarcoplasmic reticulum (SR), although changes in myofilament properties may also contribute. The present study investigated the contribution of the myofilaments to the β‐adrenergic response in isolated rat cardiac trabeculae undergoing either isometric or work‐loop contractions (involving simultaneous force generation and shortening) at different stimulation frequencies (range 0.25‐4.5 Hz). SR‐dependent effects were eliminated by treatment with ryanodine (1 μM) and cyclopiazonic acid (30 μM). In isometric contractions during SR inhibition, isoprenaline increased the force but did not alter the time course of the twitch. In contrast, in work‐loop contractions, the positive inotropic effect was accompanied by a reduced diastolic force between beats, most apparent at higher frequencies (e.g. diastolic stress fell from 58.6 ± 5.5 to 28.8 ± 5.8 mN mm−2 at 1.5 Hz). This relaxant effect contributed to a β‐adrenoceptor‐mediated increase in net work and power output at higher frequencies, by reducing the amount of work required to re‐lengthen the muscle. Consequently, the frequency for maximum power output increased from 1.1 ± 0.1 to 1.6 ± 0.1 Hz. We conclude that the contribution of myofilament properties to the relaxant effect of β‐stimulation may be of greater significance when force and length are changing simultaneously (as occurs in the heart) than during force development under isometric conditions.

Analysis of ex vivo left ventricular pressure–volume relations in the isolated murine ejecting heart

The development of microconductance technology to study cardiac pressure–volume relations in mice in vivo has significantly advanced the haemodynamic assessment of gene‐modified models of cardiovascular disease. In this study, we describe the application of microconductance analysis of cardiac function to the isolated murine ejecting heart. This ex vivo model is complementary to the previously described in vivo preparation, allows assessment without confounding effects of anaesthetic or neurohumoral influences and enables careful control of cardiac loading (particularly preload). Ex vivo pressure–volume relations in the isolated murine heart are sensitive to changes in myocardial contractility induced by β‐adrenoceptor stimulation or β‐adrenoceptor blockade, as well as the effects of chronic pressure overload induced by aortic banding. We present data for both steady‐state analyses of the Frank–Starling relation and for assessment of the left ventricular pressure–volume relation over variably loaded beats, which allows investigation of the end‐systolic and end‐diastolic pressure–volume relations. The measurement of ventricular volume in addition to pressure under carefully controlled loading conditions in the isolated ejecting heart allows a comprehensive analysis of cardiac contractile function, and provides a useful complementary model for the assessment of cardiac performance in murine models of heart disease.

Effects of α1‐ or β‐adrenoceptor stimulation on work‐loop and isometric contractions of isolated rat cardiac trabeculae

1 We studied the effects of α1−or β−adrenoceptor stimulation on the contractility of isolated rat ventricular trabeculae at 24 °C using the work‐loop technique, which simulates the cyclical changes in length and force that occur during the cardiac cycle. Some muscles were injected with fura‐2 to monitor the intracellular Ca2+ transient. 2 Comparison of twitch records revealed that peak force was greater and was reached earlier in work‐loop contractions than in corresponding isometric contractions. This was attributed to the changes in muscle length and velocity during work‐loop contractions, since the Ca2+ transients were largely unaffected by the length changes. 3 Stimulation of α1‐adrenoceptors (with 100 μM phenylephrine) increased net work, power production, the frequency for maximum work, and the frequency for maximum power production (fopt). The increase in net work was due to the positive inotropic effect of phenylephrine, which was similar at all frequencies investigated (0.33‐4.5 Hz). The increase in fopt was attributed to an abbreviation of twitch duration induced by α1‐stimulation at higher frequencies (> 1 Hz), even though the twitch became longer at 0.33 Hz. 4 β‐Adrenoceptor stimulation (with 5 μM isoprenaline) produced marked increases in net work, power output, the frequency for net work, and fopt. These effects were attributed both to the positive inotropic effect of β‐stimulation, which was greater at higher frequencies, and to the reduction in twitch duration. β‐Stimulation also abolished the frequency‐dependent acceleration of twitch duration. 5 The increase in power output and fopt with α1‐ as well as β‐adrenoceptor stimulation suggested that both receptor types may contribute to the effects of catecholamines, released during stress or exercise, although the greater effects of β‐stimulation are likely to predominate.

Molecular signalling in cardiovascular biology: from molecules to man. International symposium organized by the centre for cardiovascular biology and medicine, King's College, London.

This stimulating symposium attracted many diverse research groups, united by the common aim of understanding the molecular and cellular processes that regulate cardiovascular biology. Despite the fact that cardiovascular disease is one of the major causes of mortality worldwide, there are still many gaps in our knowledge of cardiovascular physiology and pathophysiology. Presentations at this symposium emphasized the value of the multidisciplinary approach, illustrating how findings at the cellular level have been reiterated in experimental animals and are ultimately being applied in the clinical setting. The therapeutic potential of the research presented here should become apparent in the next few years.

Molecular signalling in cardiovascular biology: from molecules to man: International Symposium Organized by the Centre for Cardiovascular Biology and Medicine, King’s College, London. King’s College, London, UK, 31 May 2000

This stimulating symposium attracted many diverse research groups, united by the common aim of understanding the molecular and cellular processes that regulate cardiovascular biology. Despite the fact that cardiovascular disease is one of the major causes of mortality worldwide, there are still many gaps in our knowledge of cardiovascular physiology and pathophysiology. Presentations at this symposium emphasized the value of the multidisciplinary approach, illustrating how findings at the cellular level have been reiterated in experimental animals and are ultimately being applied in the clinical setting. The therapeutic potential of the research presented here should become apparent in the next few years.