Sarah Calaghan - Academia.edu (original) (raw)

Papers by Sarah Calaghan

Research paper thumbnail of A Novel Pleiotropic Effect of Statins: Enhanced Cardiomyocyte β2Adrenoceptor Responsiveness

Biophysical Journal, 2010

contractile reserve decreases with consideration of protein characteristics governing the force-f... more contractile reserve decreases with consideration of protein characteristics governing the force-frequency relationship. However, the molecular alterations involved in the beta-adrenergic response lead to an increase in sensitivity. By following contractile function over time, and assessing the impact of physiologically relevant modulators of function, we will obtain a temporal resolution of cardiac function in its transition from the healthy to the diseased state.

Research paper thumbnail of Caveolae Differentially Control Phosphorylation of Sarcoplasmic Reticular Proteins Following beta2 Adrenoceptor Stimulation in the Adult Cardiac Myocyte

Biophysical Journal, 2010

myocytes, where the native Ito is small, simulation of an epicardial-level Ito accentuated the ph... more myocytes, where the native Ito is small, simulation of an epicardial-level Ito accentuated the phase 1 repolarization and significantly suppressed cell shortening by 19%. The peak amplitude of Ca2þ transient was also reduced in the presence of simulated Ito, although the rate of rise of the Ca2þ transient was increased. Conversely, subtraction, or ''blockade'' of the native Ito using the dynamic clamp enhanced contractility in epicardial cells. These results agree with the inverse correlation between Ito levels and myocyte contractility and Ca2þ transient amplitude in epicardial and endocardial myocytes. Action potential clamp and computational modeling show that phase-1 notch depth vs peak L-type influx has an inverted-U shape; shallow phase-1 notch enhances Ica-L peak, while moderate to strong phase-1 repolarization reduces Ica-L influx. Conclusion: Our results show that Ito acts as a negative, rather than positive regulator of myocyte mechanical properties in large animals.

Research paper thumbnail of The role of calcium in the response of cardiac muscle to stretch

Progress in Biophysics & Molecular Biology, 1999

Research paper thumbnail of Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes

Progress in Biophysics & Molecular Biology, 2004

The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in... more The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in several cardiac pathologies including hypertrophy and failure. In this review we describe the role that the cytoskeleton plays in modulating both the electrical activity (through ion channels and exchangers) and mechanical (or contractile) activity of the adult heart. We focus on the 3 components of the cytoskeleton, actin microfilaments, microtubules, and desmin filaments. The limited visual data available suggest that the subsarcolemmal actin cytoskeleton is sparse in the adult myocyte. Selective disruption of cytoskeletal actin by pharmacological tools has yet to be verified in the adult cell, yet evidence exists for modulation of several ionic currents, including I CaL ; I Na ; I KATP ; I SAC by actin microfilaments. Microtubules exist as a dense network throughout the adult cardiac cell, and their structure, architecture, kinetics and pharmacological manipulation are well described. Both polymerised and free tubulin are functionally significant. Microtubule proliferation reduces contraction by impeding sarcomeric motion; modulation of sarcoplasmic reticulum Ca 2þ release may also be involved in this effect. The lack of effect of microtubule disruption on cardiac contractility in adult myocytes, and the concentration-dependent modulation of the rate of contraction by the disruptor nocodazole in neonatal myocytes, support the existence of functionally distinct microtubule populations. We address the controversy regarding the stimulation of the b-adrenergic signalling pathway by free tubulin. Work with mice lacking desmin has demonstrated the importance of intermediate filaments to normal cardiac function, but the precise role that desmin plays in the electrical and mechanical activity of cardiac muscle has yet to be determined. r

Research paper thumbnail of Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca 2+ and contraction following β-adrenergic stimulation of rat heart

Pflugers Archiv-european Journal of Physiology, 1998

Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phosphola... more Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phospholamban, the intracellular calcium ([Ca2+]i) transient and contraction were measured in isolated rat ventricular myocytes exposed to the β-adrenoceptor agonist isoprenaline. Cyclic AMP was measured by [125I]-cAMP scintillation proximity assay, phosphorylation of phospholamban at Ser16 and Thr17 was assessed using a pair of site-specific polyclonal antibodies, and [Ca2+]i was monitored with the fluorescent dye fura 2. Cyclic AMP rose to twice basal levels in the presence of 10–6 M isoprenaline. The maximum increase in phosphorylation at Ser16 and Thr17 of phospholamban was seen at 10–7 M isoprenaline. At this stage Ser16 phosphorylation was six times higher, and Thr17 phosphorylation was three times higher than that recorded in the absence of isoprenaline. Phosphorylation at Ser16 correlated more closely with changes in the [Ca2+]i transient and contraction than did phosphorylation at Thr17. This is the first study of its kind to measure simultaneous changes in cAMP, the phosphorylation of phospholamban, the [Ca2+]i transient and contraction over a range of concentrations of β-agonist. The results suggest that phosphorylation of phospholamban at Thr17 is of lesser physiological relevance to the effects of β-adrenergic stimulation on the heart than phosphorylation at Ser16.

Research paper thumbnail of Caveolae Act As Membrane Reserves Which May Limit I Cl,swell Activation During Cardiac Myocyte Swelling

Biophysical Journal, 2009

Bacterial mechanosensitive (MS) channels protect bacterial cells from osmotic shock, acting as em... more Bacterial mechanosensitive (MS) channels protect bacterial cells from osmotic shock, acting as emergency relief valves (1,2). E. coli has three such channels, the MS channel of large conductance (MscL), the MS channel of small conductance (MscS) and the MS channel of mini conductance (MscM). Both MscL and MscS have been extensively studied using the patch-clamp technique in giant spheroplasts . However, only MscL incorporates efficiently in liposomes (2,5). Here we report the first example of co-reconstitution of both MscS and MscL into azolectin liposomes. We also report reconstitution efficiencies of both proteins into liposomes of different lipid composition and using different incorporation methods.

Research paper thumbnail of Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte

Journal of Molecular and Cellular Cardiology, 2008

Cyclic AMP exhibits local (sarcolemmal) and global (cytosolic) patterns of signalling, allowing r... more Cyclic AMP exhibits local (sarcolemmal) and global (cytosolic) patterns of signalling, allowing receptor-specific signals to be generated by a single second messenger. Here we determine whether caveolae, invaginated lipid rafts, are responsible for confining the β 2 adrenoceptor (AR) cAMP signal to the sarcolemmal compartment. Myocytes were treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MβC) to disrupt caveolae. Caveolae-containing membrane fractions were isolated by detergent-free sucrose gradient fractionation. Cell shortening and phosphorylation of the sarcoplasmic reticular protein phospholamban (PLB) and the myofilament protein troponin I (TnI) were measured in response to β 2 AR stimulation (with salbutamol in the presence of 1 μM atenolol). Ser 16 phosphorylation of PLB (pPLB), Ser 22,23 phosphorylation of TnI (pTnI), and positive lusitropy were used as indices of global cAMP signals. The ability of MβC to disrupt caveolae was confirmed by selective depletion of the buoyant membrane fractions of cholesterol and caveolin 3, the 2 essential components of caveolae. In control cells, no change in pPLB, pTnI or time to half relaxation was recorded with β 2 AR stimulation (P N 0.05), but following caveolar disruption a 60-70% increase in phosphorylation of both proteins was seen, accompanied by positive lusitropy (P b 0.05). These data show for the first time that disrupting caveolae converts the sarcolemmal-confined cAMP signal associated with β 2 AR stimulation to a global signal that targets proteins of the sarcoplasmic reticulum and myofilaments, with functional sequelae. The role of caveolae in spatial control of cAMP may be relevant to perturbation of β AR signalling in cardiovascular disease.

Research paper thumbnail of Effects of Cholesterol Depletion on Compartmentalized cAMP Responses in Adult Cardiac Myocytes

Biophysical Journal, 2011

β 1 -Adrenergic receptors (β 1 ARs) and E-type prostaglandin receptors (EPRs) both produce compar... more β 1 -Adrenergic receptors (β 1 ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β 1 ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β 1 AR activation enhanced the Ltype Ca 2+ current, intracellular Ca 2+ transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β 1 ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β 1 AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β 1 AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β 1 AR and EPR responses. They also suggest that β 1 AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

Research paper thumbnail of Heterologous expression of wild-type and mutant beta-cardiac myosin changes the contractile kinetics of cultured mouse myotubes

Journal of Physiology-london, 2003

The properties of myosin expressed in muscle are a major determinant of muscle performance. In th... more The properties of myosin expressed in muscle are a major determinant of muscle performance. In this study we used a novel approach to examine the functional impact of changes in myosin heavy chain (MHC) isoform expression, as well as the consequences of expressing the mutant MHC implicated in familial hypertrophic cardiomyopathy (FHC). Cultured mouse myoblasts that normally express fast embryonic myosin were untransfected, or stably transfected with a plasmid expressing either wild-type (cWT) or mutant (D778G or G741R) b-cardiac myosin. After differentiation for 5-7 days, cWT or mutant b-cardiac myosin was expressed at 25 % of total myosin in the myotube. We measured time-to-peak shortening (ttp), time for half-relaxation (t 0.5 ), the maximum velocity of shortening (V max ) at 1 Hz stimulation, and the tetanic fusion frequency. Expression of cWT b-cardiac myosin significantly increased ttp and t 0.5 and decreased the fusion frequency compared with untransfected myotubes. However, when we compared myotubes expressing mutant b-cardiac myosin with those expressing cWT b-cardiac myosin, we found that ttp and t 0.5 were significantly decreased, and V max was increased for the D778G mutant, whereas ttp, t 0.5 and V max were unchanged for the G741R mutant. The fusion frequency was increased for both mutant myosins. Our data support the conclusion that the impact of the slower myosin isoform dominates when both slow and fast isoforms are present. This work suggests that FHC associated with either D778G or G741R mutation in MHC is an 'energy cost' disease, but that the phenotype of D778G is more severe than that of G741R.

Research paper thumbnail of Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes

Journal of Molecular and Cellular Cardiology, 2011

β 1 -Adrenergic receptors (β 1 ARs) and E-type prostaglandin receptors (EPRs) both produce compar... more β 1 -Adrenergic receptors (β 1 ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β 1 ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β 1 AR activation enhanced the Ltype Ca 2+ current, intracellular Ca 2+ transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β 1 ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β 1 AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β 1 AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β 1 AR and EPR responses. They also suggest that β 1 AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

Research paper thumbnail of Caveolae compartmentalise β2-adrenoceptor signals by curtailing cAMP production and maintaining phosphatase activity in the sarcoplasmic reticulum of the adult ventricular myocyte

Journal of Molecular and Cellular Cardiology

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-... more Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca(2+) current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser(16). Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Research paper thumbnail of Activation of Na+-H+ exchange and stretch-activated channels underlies the slow inotropic response to stretch in myocytes and muscle from the rat heart

Journal of Physiology-london, 2004

We present the first direct comparison of the major candidates proposed to underlie the slow phas... more We present the first direct comparison of the major candidates proposed to underlie the slow phase of the force increase seen following myocardial stretch: (i) the Na + -H + exchanger (NHE) (ii) nitric oxide (NO) and the ryanodine receptor (RyR) and (iii) the stretch-activated channel (SAC) in both single myocytes and multicellular muscle preparations from the rat heart. Ventricular myocytes were stretched by approximately 7% using carbon fibres. Papillary muscles were stretched from 88 to 98% of the length at which maximum tension is generated (L max ). Inhibition of NHE with HOE 642 (5 µM) significantly reduced (P < 0.05) the magnitude of the slow force response in both muscle and myocytes. Neither inhibition of phosphatidylinositol-3-OH kinase (PtdIns-3-OH kinase) with LY294002 (10 µM) nor NO synthase with L-NAME (1 mM) reduced the slow force response in muscle or myocytes (P > 0.05), and the slow response was still present in the single myocyte when the sarcoplasmic reticulum was rigorously inhibited with 1 µM ryanodine and 1 µM thapsigargin. We saw a significant reduction (P < 0.05) in the slow force response in the presence of the SAC blocker streptomycin in both muscle (80 µM) and myocytes (40 µM). In fura 2-loaded myocytes, HOE 642 and streptomycin, but not L-NAME, ablated the stretch-induced increase in [Ca 2+ ] i transient amplitude. Our data suggest that in the rat, under our experimental conditions, there are two mechanisms that underlie the slow inotropic response to stretch: activation of NHE; and of activation of SACs. Both these mechanisms are intrinsic to the myocyte.

Research paper thumbnail of Contribution of angiotensin II, endothelin 1 and the endothelium to the slow inotropic response to stretch in ferret papillary muscle

Pflugers Archiv-european Journal of Physiology, 2001

We investigated the contribution of angiotensin II and endothelin 1 to the slow positive inotropi... more We investigated the contribution of angiotensin II and endothelin 1 to the slow positive inotropic response observed following stretch of isolated ferret papillary muscle from 88% to 98% of the length at which maximum force is generated. Angiotensin antagonists losartan and saralasin did not affect the magnitude of the slow response in ferret papillary muscle. The ETA-selective antagonist BQ123 slightly reduced the magnitude of the slow response (P>0.05). In the presence of PD145065 (an ETA and ETB antagonist), the magnitude of the slow response was reduced significantly by 50%. Removal of the endothelium with 1% Triton X-100 reversed the slow response to stretch. We conclude that, in the ferret, endothelin 1 acting through ETA and ETB receptors, contributes to the slow response although it is not the sole mediator. Angiotensin II is not a prerequisite for the slow response to stretch. We have shown for the first time that the endocardial endothelium plays a pivotal role in this phenomenon in cardiac papillary muscle.

Research paper thumbnail of Do stretch-induced changes in intracellular calcium modify the electrical activity of cardiac muscle

Progress in Biophysics & Molecular Biology, 2003

Stretch of the myocardium influences the shape and amplitude of the intracellular Ca 2+ ([Ca 2+ ]... more Stretch of the myocardium influences the shape and amplitude of the intracellular Ca 2+ ([Ca 2+ ] i ) transient. Under isometric conditions stretch immediately increases myofilament Ca 2+ sensitivity, increasing force production and abbreviating the time course of the [Ca 2+ ] i transient (the rapid response). Conversely, muscle shortening can prolong the Ca 2+ transient by decreasing myofilament Ca 2+ sensitivity. During the cardiac cycle, increased ventricular dilation may increase myofilament Ca 2+ sensitivity during diastolic filling and the isovolumic phase of systole, but enhance the decrease in myofilament Ca 2+ sensitivity during the systolic shortening of the ejection phase. If stretch is maintained there is a gradual increase in the amplitude of the Ca 2+ transient and force production, which takes several minutes to develop fully (the slow response). The rapid and slow responses have been reported in whole hearts and single myocytes. Here we review stretch-induced changes in [Ca 2+ ] i and the underlying mechanisms.

Research paper thumbnail of Caveolae Act as Membrane Reserves Which Limit Mechanosensitive ICl,swell Channel Activation during Swelling in the Rat Ventricular Myocyte

PLOS One, 2009

Background: Many ion channels are preferentially located in caveolae where compartmentalisation/s... more Background: Many ion channels are preferentially located in caveolae where compartmentalisation/scaffolding with signal transduction components regulates their activity. Channels that are mechanosensitive may be additionally dependent on caveolar control of the mechanical state of the membrane. Here we test which mechanism underlies caveolar-regulation of the mechanosensitive I Cl,swell channel in the adult cardiac myocyte.

Research paper thumbnail of Caveolae Act as Membrane Reserves Which Limit Mechanosensitive ICl,swell Channel Activation during Swelling in the Rat Ventricular Myocyte

PLOS One, 2009

Background: Many ion channels are preferentially located in caveolae where compartmentalisation/s... more Background: Many ion channels are preferentially located in caveolae where compartmentalisation/scaffolding with signal transduction components regulates their activity. Channels that are mechanosensitive may be additionally dependent on caveolar control of the mechanical state of the membrane. Here we test which mechanism underlies caveolar-regulation of the mechanosensitive I Cl,swell channel in the adult cardiac myocyte.

Research paper thumbnail of Cyclic AMP but not phosphorylation of phospholamban contributes to the slow inotropic response to stretch in ferret papillary muscle

Pflugers Archiv-european Journal of Physiology, 1999

cAMP has been suggested to mediate the increased intracellular Ca2+ transient and contraction se... more cAMP has been suggested to mediate the increased intracellular Ca2+ transient and contraction seen during the slow response to stretch in cardiac muscle. We measured cAMP in ferret papillary muscles stretched from 80–85% to 98% of their length at which maximum active tension is produced (L max) for 15 min. cAMP was significantly (P<0.05) increased by 53% in muscles at the longer length which showed the slow response compared with controls. By contrast, in a population of muscles that were stretched but did not show the slow response, cAMP was not significantly different from that in muscles at the short length. Although cAMP can increase sarcoplasmic reticulum (SR) Ca2+ uptake by phosphorylation of phospholamban, we found no significant effect of stretch on phosphorylation of phospholamban at either Ser16 or Thr17. Further support for the hypothesis that cAMP is a mediator of the slow response was obtained by exposure of some muscles to the cell-permeable cAMP antagonist 8-bromo, adenosine 3′,5′-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMPS, (2.5–10 µM). The slow response was reduced by 30% (P<0.05) in the presence of this antagonist. Our results not only provide evidence for the mediation of the slow response to stretch by cAMP, they also suggest that cAMP may rise in an intracellular compartment inaccessible to the SR.

Research paper thumbnail of Stable microtubules contribute to cardiac dysfunction in the streptozotocin-induced model of type 1 diabetes in the rat

Molecular and Cellular Biochemistry, 2007

Cardiac microtubule stability is increased in the streptozotocin (STZ) model of type 1 diabetes. ... more Cardiac microtubule stability is increased in the streptozotocin (STZ) model of type 1 diabetes. Here, we investigate the reason for increased microtubule stability, and the functional consequences of stable microtubule disruption. Ventricular myocytes were isolated from rats at 8–12 weeks after injection of STZ. A 10% increase in microtubule density, but no difference in the ratio of microtubule-associated protein 4 (MAP4) to tubulin was seen in myocytes from STZ rats. Functionally, STZ myocytes showed a tendency for reduced shortening and intracellular Ca2+ ([Ca2+]i ) transient amplitude, and a significant prolongation of time to peak (ttp) shortening and [Ca2+]i . Although microtubules in STZ myocytes were less sensitive to the microtubule disruptor nocodazole (NOC; 33 μM) than control myocytes, we only saw marked functional consequences of microtubule disruption by NOC in myocytes from diabetic animals. NOC increased shortening and [Ca2+]i transient amplitude in STZ myocytes by 45 and 24%, respectively (compared with 4 and 6% in controls). Likewise, NOC decreased ttp shortening and [Ca2+]i only in STZ myocytes, such that these parameters were no longer different between the two groups. In conclusion, stable microtubules in diabetes are not associated with an increase in MAP4, but are functionally relevant to cardiac dysfunction in diabetes, regulating both [Ca2+]i and shortening.

Research paper thumbnail of Preservation of the in VivoPhosphorylation Status of Phospholamban in the Heart: Evidence for a Site-Specific Difference in the Dephosphorylation of Phospholamban

Biochemical and Biophysical Research Communications, 1998

The phosphorylation status of the cardiac sarcoplasis removed and SR Ca 2/ uptake is increased (1... more The phosphorylation status of the cardiac sarcoplasis removed and SR Ca 2/ uptake is increased (1). In mic reticular (SR) protein phospholamban determines vivo, phosphorylation takes place at Ser 16 and Thr 17 by the activity of the SR Ca 2/ -ATPase. In order to predict cAMP-dependent protein kinase and Ca 2/ /calmodulin-SR Ca 2/ transport in vivo, it is vital that techniques dependent protein kinase respectively (2). Endogenous used to measure the phosphorylation status of phosphosphatases act to reverse the action of these kinases pholamban adequately clamp the endogenous kinases (3). The phosphorylation status of phospholamban is and phosphatases which modify phosphorylation durmodified primarily by b-adrenergic stimulation, which ing sample preparation. A recent study (Boateng, S., increases phosphorylation at both sites , but changes Seymour, A-M., Dunn, M., Yacoub, M., and Boheler, K. in phosphorylation have also been observed in patho-(1997) Biochem. Biophys. Res. Comm. 239, 701-705) has

Research paper thumbnail of The Role of the Sarcomere and Cytoskeleton in Cardiac Mechanotransduction

The basic contractile unit of the cardiac myocyte is the sarcomere. Force develops as a result of... more The basic contractile unit of the cardiac myocyte is the sarcomere. Force develops as a result of the interaction of myosin heads with the actin thin filament. Actin filaments are directly connected to the Z line of the sarcomere, whereas myosin filaments are secured via the giant elastic protein titin. When cardiac muscle is stretched there is an immediate increase in contractility. This is an acute and fundamental cardiac adaptive response to an increase in demand. Evidence suggests that an increase in the probability of crossbridge formation, through titin strain and positive cooperative mechanisms, underlies the length-dependent activation of cardiac muscle. The sarcomere is connected to the sarcolemma by cytoskeletal components which link the Z-line with the membrane-spanning integrins and dystroglycan complex. Integrins and dystroglycan, in turn, bind to components of the extracellular matrix, such as laminin, which sheath the cardiac myocyte. Connections also exist between Z-line and nucleus via the intermediate filament protein desmin. The intracellular connections between the Z-line of the sarcomere and the sarcolemma allow transmission of force developed by the myofilaments. However, the physical pathway that links the extracellular matrix, membrane-spanning proteins, and the cell interior also plays a fundamental role in mechanotransduction. These links allow the cell to sense and respond to mechanical stimuli through connections with the cytoskeleton and activation of signalling cascades.

Research paper thumbnail of A Novel Pleiotropic Effect of Statins: Enhanced Cardiomyocyte β2Adrenoceptor Responsiveness

Biophysical Journal, 2010

contractile reserve decreases with consideration of protein characteristics governing the force-f... more contractile reserve decreases with consideration of protein characteristics governing the force-frequency relationship. However, the molecular alterations involved in the beta-adrenergic response lead to an increase in sensitivity. By following contractile function over time, and assessing the impact of physiologically relevant modulators of function, we will obtain a temporal resolution of cardiac function in its transition from the healthy to the diseased state.

Research paper thumbnail of Caveolae Differentially Control Phosphorylation of Sarcoplasmic Reticular Proteins Following beta2 Adrenoceptor Stimulation in the Adult Cardiac Myocyte

Biophysical Journal, 2010

myocytes, where the native Ito is small, simulation of an epicardial-level Ito accentuated the ph... more myocytes, where the native Ito is small, simulation of an epicardial-level Ito accentuated the phase 1 repolarization and significantly suppressed cell shortening by 19%. The peak amplitude of Ca2þ transient was also reduced in the presence of simulated Ito, although the rate of rise of the Ca2þ transient was increased. Conversely, subtraction, or ''blockade'' of the native Ito using the dynamic clamp enhanced contractility in epicardial cells. These results agree with the inverse correlation between Ito levels and myocyte contractility and Ca2þ transient amplitude in epicardial and endocardial myocytes. Action potential clamp and computational modeling show that phase-1 notch depth vs peak L-type influx has an inverted-U shape; shallow phase-1 notch enhances Ica-L peak, while moderate to strong phase-1 repolarization reduces Ica-L influx. Conclusion: Our results show that Ito acts as a negative, rather than positive regulator of myocyte mechanical properties in large animals.

Research paper thumbnail of The role of calcium in the response of cardiac muscle to stretch

Progress in Biophysics & Molecular Biology, 1999

Research paper thumbnail of Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes

Progress in Biophysics & Molecular Biology, 2004

The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in... more The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in several cardiac pathologies including hypertrophy and failure. In this review we describe the role that the cytoskeleton plays in modulating both the electrical activity (through ion channels and exchangers) and mechanical (or contractile) activity of the adult heart. We focus on the 3 components of the cytoskeleton, actin microfilaments, microtubules, and desmin filaments. The limited visual data available suggest that the subsarcolemmal actin cytoskeleton is sparse in the adult myocyte. Selective disruption of cytoskeletal actin by pharmacological tools has yet to be verified in the adult cell, yet evidence exists for modulation of several ionic currents, including I CaL ; I Na ; I KATP ; I SAC by actin microfilaments. Microtubules exist as a dense network throughout the adult cardiac cell, and their structure, architecture, kinetics and pharmacological manipulation are well described. Both polymerised and free tubulin are functionally significant. Microtubule proliferation reduces contraction by impeding sarcomeric motion; modulation of sarcoplasmic reticulum Ca 2þ release may also be involved in this effect. The lack of effect of microtubule disruption on cardiac contractility in adult myocytes, and the concentration-dependent modulation of the rate of contraction by the disruptor nocodazole in neonatal myocytes, support the existence of functionally distinct microtubule populations. We address the controversy regarding the stimulation of the b-adrenergic signalling pathway by free tubulin. Work with mice lacking desmin has demonstrated the importance of intermediate filaments to normal cardiac function, but the precise role that desmin plays in the electrical and mechanical activity of cardiac muscle has yet to be determined. r

Research paper thumbnail of Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca 2+ and contraction following β-adrenergic stimulation of rat heart

Pflugers Archiv-european Journal of Physiology, 1998

Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phosphola... more Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phospholamban, the intracellular calcium ([Ca2+]i) transient and contraction were measured in isolated rat ventricular myocytes exposed to the β-adrenoceptor agonist isoprenaline. Cyclic AMP was measured by [125I]-cAMP scintillation proximity assay, phosphorylation of phospholamban at Ser16 and Thr17 was assessed using a pair of site-specific polyclonal antibodies, and [Ca2+]i was monitored with the fluorescent dye fura 2. Cyclic AMP rose to twice basal levels in the presence of 10–6 M isoprenaline. The maximum increase in phosphorylation at Ser16 and Thr17 of phospholamban was seen at 10–7 M isoprenaline. At this stage Ser16 phosphorylation was six times higher, and Thr17 phosphorylation was three times higher than that recorded in the absence of isoprenaline. Phosphorylation at Ser16 correlated more closely with changes in the [Ca2+]i transient and contraction than did phosphorylation at Thr17. This is the first study of its kind to measure simultaneous changes in cAMP, the phosphorylation of phospholamban, the [Ca2+]i transient and contraction over a range of concentrations of β-agonist. The results suggest that phosphorylation of phospholamban at Thr17 is of lesser physiological relevance to the effects of β-adrenergic stimulation on the heart than phosphorylation at Ser16.

Research paper thumbnail of Caveolae Act As Membrane Reserves Which May Limit I Cl,swell Activation During Cardiac Myocyte Swelling

Biophysical Journal, 2009

Bacterial mechanosensitive (MS) channels protect bacterial cells from osmotic shock, acting as em... more Bacterial mechanosensitive (MS) channels protect bacterial cells from osmotic shock, acting as emergency relief valves (1,2). E. coli has three such channels, the MS channel of large conductance (MscL), the MS channel of small conductance (MscS) and the MS channel of mini conductance (MscM). Both MscL and MscS have been extensively studied using the patch-clamp technique in giant spheroplasts . However, only MscL incorporates efficiently in liposomes (2,5). Here we report the first example of co-reconstitution of both MscS and MscL into azolectin liposomes. We also report reconstitution efficiencies of both proteins into liposomes of different lipid composition and using different incorporation methods.

Research paper thumbnail of Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte

Journal of Molecular and Cellular Cardiology, 2008

Cyclic AMP exhibits local (sarcolemmal) and global (cytosolic) patterns of signalling, allowing r... more Cyclic AMP exhibits local (sarcolemmal) and global (cytosolic) patterns of signalling, allowing receptor-specific signals to be generated by a single second messenger. Here we determine whether caveolae, invaginated lipid rafts, are responsible for confining the β 2 adrenoceptor (AR) cAMP signal to the sarcolemmal compartment. Myocytes were treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MβC) to disrupt caveolae. Caveolae-containing membrane fractions were isolated by detergent-free sucrose gradient fractionation. Cell shortening and phosphorylation of the sarcoplasmic reticular protein phospholamban (PLB) and the myofilament protein troponin I (TnI) were measured in response to β 2 AR stimulation (with salbutamol in the presence of 1 μM atenolol). Ser 16 phosphorylation of PLB (pPLB), Ser 22,23 phosphorylation of TnI (pTnI), and positive lusitropy were used as indices of global cAMP signals. The ability of MβC to disrupt caveolae was confirmed by selective depletion of the buoyant membrane fractions of cholesterol and caveolin 3, the 2 essential components of caveolae. In control cells, no change in pPLB, pTnI or time to half relaxation was recorded with β 2 AR stimulation (P N 0.05), but following caveolar disruption a 60-70% increase in phosphorylation of both proteins was seen, accompanied by positive lusitropy (P b 0.05). These data show for the first time that disrupting caveolae converts the sarcolemmal-confined cAMP signal associated with β 2 AR stimulation to a global signal that targets proteins of the sarcoplasmic reticulum and myofilaments, with functional sequelae. The role of caveolae in spatial control of cAMP may be relevant to perturbation of β AR signalling in cardiovascular disease.

Research paper thumbnail of Effects of Cholesterol Depletion on Compartmentalized cAMP Responses in Adult Cardiac Myocytes

Biophysical Journal, 2011

β 1 -Adrenergic receptors (β 1 ARs) and E-type prostaglandin receptors (EPRs) both produce compar... more β 1 -Adrenergic receptors (β 1 ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β 1 ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β 1 AR activation enhanced the Ltype Ca 2+ current, intracellular Ca 2+ transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β 1 ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β 1 AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β 1 AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β 1 AR and EPR responses. They also suggest that β 1 AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

Research paper thumbnail of Heterologous expression of wild-type and mutant beta-cardiac myosin changes the contractile kinetics of cultured mouse myotubes

Journal of Physiology-london, 2003

The properties of myosin expressed in muscle are a major determinant of muscle performance. In th... more The properties of myosin expressed in muscle are a major determinant of muscle performance. In this study we used a novel approach to examine the functional impact of changes in myosin heavy chain (MHC) isoform expression, as well as the consequences of expressing the mutant MHC implicated in familial hypertrophic cardiomyopathy (FHC). Cultured mouse myoblasts that normally express fast embryonic myosin were untransfected, or stably transfected with a plasmid expressing either wild-type (cWT) or mutant (D778G or G741R) b-cardiac myosin. After differentiation for 5-7 days, cWT or mutant b-cardiac myosin was expressed at 25 % of total myosin in the myotube. We measured time-to-peak shortening (ttp), time for half-relaxation (t 0.5 ), the maximum velocity of shortening (V max ) at 1 Hz stimulation, and the tetanic fusion frequency. Expression of cWT b-cardiac myosin significantly increased ttp and t 0.5 and decreased the fusion frequency compared with untransfected myotubes. However, when we compared myotubes expressing mutant b-cardiac myosin with those expressing cWT b-cardiac myosin, we found that ttp and t 0.5 were significantly decreased, and V max was increased for the D778G mutant, whereas ttp, t 0.5 and V max were unchanged for the G741R mutant. The fusion frequency was increased for both mutant myosins. Our data support the conclusion that the impact of the slower myosin isoform dominates when both slow and fast isoforms are present. This work suggests that FHC associated with either D778G or G741R mutation in MHC is an 'energy cost' disease, but that the phenotype of D778G is more severe than that of G741R.

Research paper thumbnail of Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes

Journal of Molecular and Cellular Cardiology, 2011

β 1 -Adrenergic receptors (β 1 ARs) and E-type prostaglandin receptors (EPRs) both produce compar... more β 1 -Adrenergic receptors (β 1 ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β 1 ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β 1 AR activation enhanced the Ltype Ca 2+ current, intracellular Ca 2+ transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β 1 ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β 1 AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β 1 AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β 1 AR and EPR responses. They also suggest that β 1 AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

Research paper thumbnail of Caveolae compartmentalise β2-adrenoceptor signals by curtailing cAMP production and maintaining phosphatase activity in the sarcoplasmic reticulum of the adult ventricular myocyte

Journal of Molecular and Cellular Cardiology

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-... more Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca(2+) current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser(16). Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;Local Signaling in Myocytes&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;.

Research paper thumbnail of Activation of Na+-H+ exchange and stretch-activated channels underlies the slow inotropic response to stretch in myocytes and muscle from the rat heart

Journal of Physiology-london, 2004

We present the first direct comparison of the major candidates proposed to underlie the slow phas... more We present the first direct comparison of the major candidates proposed to underlie the slow phase of the force increase seen following myocardial stretch: (i) the Na + -H + exchanger (NHE) (ii) nitric oxide (NO) and the ryanodine receptor (RyR) and (iii) the stretch-activated channel (SAC) in both single myocytes and multicellular muscle preparations from the rat heart. Ventricular myocytes were stretched by approximately 7% using carbon fibres. Papillary muscles were stretched from 88 to 98% of the length at which maximum tension is generated (L max ). Inhibition of NHE with HOE 642 (5 µM) significantly reduced (P < 0.05) the magnitude of the slow force response in both muscle and myocytes. Neither inhibition of phosphatidylinositol-3-OH kinase (PtdIns-3-OH kinase) with LY294002 (10 µM) nor NO synthase with L-NAME (1 mM) reduced the slow force response in muscle or myocytes (P > 0.05), and the slow response was still present in the single myocyte when the sarcoplasmic reticulum was rigorously inhibited with 1 µM ryanodine and 1 µM thapsigargin. We saw a significant reduction (P < 0.05) in the slow force response in the presence of the SAC blocker streptomycin in both muscle (80 µM) and myocytes (40 µM). In fura 2-loaded myocytes, HOE 642 and streptomycin, but not L-NAME, ablated the stretch-induced increase in [Ca 2+ ] i transient amplitude. Our data suggest that in the rat, under our experimental conditions, there are two mechanisms that underlie the slow inotropic response to stretch: activation of NHE; and of activation of SACs. Both these mechanisms are intrinsic to the myocyte.

Research paper thumbnail of Contribution of angiotensin II, endothelin 1 and the endothelium to the slow inotropic response to stretch in ferret papillary muscle

Pflugers Archiv-european Journal of Physiology, 2001

We investigated the contribution of angiotensin II and endothelin 1 to the slow positive inotropi... more We investigated the contribution of angiotensin II and endothelin 1 to the slow positive inotropic response observed following stretch of isolated ferret papillary muscle from 88% to 98% of the length at which maximum force is generated. Angiotensin antagonists losartan and saralasin did not affect the magnitude of the slow response in ferret papillary muscle. The ETA-selective antagonist BQ123 slightly reduced the magnitude of the slow response (P>0.05). In the presence of PD145065 (an ETA and ETB antagonist), the magnitude of the slow response was reduced significantly by 50%. Removal of the endothelium with 1% Triton X-100 reversed the slow response to stretch. We conclude that, in the ferret, endothelin 1 acting through ETA and ETB receptors, contributes to the slow response although it is not the sole mediator. Angiotensin II is not a prerequisite for the slow response to stretch. We have shown for the first time that the endocardial endothelium plays a pivotal role in this phenomenon in cardiac papillary muscle.

Research paper thumbnail of Do stretch-induced changes in intracellular calcium modify the electrical activity of cardiac muscle

Progress in Biophysics & Molecular Biology, 2003

Stretch of the myocardium influences the shape and amplitude of the intracellular Ca 2+ ([Ca 2+ ]... more Stretch of the myocardium influences the shape and amplitude of the intracellular Ca 2+ ([Ca 2+ ] i ) transient. Under isometric conditions stretch immediately increases myofilament Ca 2+ sensitivity, increasing force production and abbreviating the time course of the [Ca 2+ ] i transient (the rapid response). Conversely, muscle shortening can prolong the Ca 2+ transient by decreasing myofilament Ca 2+ sensitivity. During the cardiac cycle, increased ventricular dilation may increase myofilament Ca 2+ sensitivity during diastolic filling and the isovolumic phase of systole, but enhance the decrease in myofilament Ca 2+ sensitivity during the systolic shortening of the ejection phase. If stretch is maintained there is a gradual increase in the amplitude of the Ca 2+ transient and force production, which takes several minutes to develop fully (the slow response). The rapid and slow responses have been reported in whole hearts and single myocytes. Here we review stretch-induced changes in [Ca 2+ ] i and the underlying mechanisms.

Research paper thumbnail of Caveolae Act as Membrane Reserves Which Limit Mechanosensitive ICl,swell Channel Activation during Swelling in the Rat Ventricular Myocyte

PLOS One, 2009

Background: Many ion channels are preferentially located in caveolae where compartmentalisation/s... more Background: Many ion channels are preferentially located in caveolae where compartmentalisation/scaffolding with signal transduction components regulates their activity. Channels that are mechanosensitive may be additionally dependent on caveolar control of the mechanical state of the membrane. Here we test which mechanism underlies caveolar-regulation of the mechanosensitive I Cl,swell channel in the adult cardiac myocyte.

Research paper thumbnail of Caveolae Act as Membrane Reserves Which Limit Mechanosensitive ICl,swell Channel Activation during Swelling in the Rat Ventricular Myocyte

PLOS One, 2009

Background: Many ion channels are preferentially located in caveolae where compartmentalisation/s... more Background: Many ion channels are preferentially located in caveolae where compartmentalisation/scaffolding with signal transduction components regulates their activity. Channels that are mechanosensitive may be additionally dependent on caveolar control of the mechanical state of the membrane. Here we test which mechanism underlies caveolar-regulation of the mechanosensitive I Cl,swell channel in the adult cardiac myocyte.

Research paper thumbnail of Cyclic AMP but not phosphorylation of phospholamban contributes to the slow inotropic response to stretch in ferret papillary muscle

Pflugers Archiv-european Journal of Physiology, 1999

cAMP has been suggested to mediate the increased intracellular Ca2+ transient and contraction se... more cAMP has been suggested to mediate the increased intracellular Ca2+ transient and contraction seen during the slow response to stretch in cardiac muscle. We measured cAMP in ferret papillary muscles stretched from 80–85% to 98% of their length at which maximum active tension is produced (L max) for 15 min. cAMP was significantly (P<0.05) increased by 53% in muscles at the longer length which showed the slow response compared with controls. By contrast, in a population of muscles that were stretched but did not show the slow response, cAMP was not significantly different from that in muscles at the short length. Although cAMP can increase sarcoplasmic reticulum (SR) Ca2+ uptake by phosphorylation of phospholamban, we found no significant effect of stretch on phosphorylation of phospholamban at either Ser16 or Thr17. Further support for the hypothesis that cAMP is a mediator of the slow response was obtained by exposure of some muscles to the cell-permeable cAMP antagonist 8-bromo, adenosine 3′,5′-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMPS, (2.5–10 µM). The slow response was reduced by 30% (P<0.05) in the presence of this antagonist. Our results not only provide evidence for the mediation of the slow response to stretch by cAMP, they also suggest that cAMP may rise in an intracellular compartment inaccessible to the SR.

Research paper thumbnail of Stable microtubules contribute to cardiac dysfunction in the streptozotocin-induced model of type 1 diabetes in the rat

Molecular and Cellular Biochemistry, 2007

Cardiac microtubule stability is increased in the streptozotocin (STZ) model of type 1 diabetes. ... more Cardiac microtubule stability is increased in the streptozotocin (STZ) model of type 1 diabetes. Here, we investigate the reason for increased microtubule stability, and the functional consequences of stable microtubule disruption. Ventricular myocytes were isolated from rats at 8–12 weeks after injection of STZ. A 10% increase in microtubule density, but no difference in the ratio of microtubule-associated protein 4 (MAP4) to tubulin was seen in myocytes from STZ rats. Functionally, STZ myocytes showed a tendency for reduced shortening and intracellular Ca2+ ([Ca2+]i ) transient amplitude, and a significant prolongation of time to peak (ttp) shortening and [Ca2+]i . Although microtubules in STZ myocytes were less sensitive to the microtubule disruptor nocodazole (NOC; 33 μM) than control myocytes, we only saw marked functional consequences of microtubule disruption by NOC in myocytes from diabetic animals. NOC increased shortening and [Ca2+]i transient amplitude in STZ myocytes by 45 and 24%, respectively (compared with 4 and 6% in controls). Likewise, NOC decreased ttp shortening and [Ca2+]i only in STZ myocytes, such that these parameters were no longer different between the two groups. In conclusion, stable microtubules in diabetes are not associated with an increase in MAP4, but are functionally relevant to cardiac dysfunction in diabetes, regulating both [Ca2+]i and shortening.

Research paper thumbnail of Preservation of the in VivoPhosphorylation Status of Phospholamban in the Heart: Evidence for a Site-Specific Difference in the Dephosphorylation of Phospholamban

Biochemical and Biophysical Research Communications, 1998

The phosphorylation status of the cardiac sarcoplasis removed and SR Ca 2/ uptake is increased (1... more The phosphorylation status of the cardiac sarcoplasis removed and SR Ca 2/ uptake is increased (1). In mic reticular (SR) protein phospholamban determines vivo, phosphorylation takes place at Ser 16 and Thr 17 by the activity of the SR Ca 2/ -ATPase. In order to predict cAMP-dependent protein kinase and Ca 2/ /calmodulin-SR Ca 2/ transport in vivo, it is vital that techniques dependent protein kinase respectively (2). Endogenous used to measure the phosphorylation status of phosphosphatases act to reverse the action of these kinases pholamban adequately clamp the endogenous kinases (3). The phosphorylation status of phospholamban is and phosphatases which modify phosphorylation durmodified primarily by b-adrenergic stimulation, which ing sample preparation. A recent study (Boateng, S., increases phosphorylation at both sites , but changes Seymour, A-M., Dunn, M., Yacoub, M., and Boheler, K. in phosphorylation have also been observed in patho-(1997) Biochem. Biophys. Res. Comm. 239, 701-705) has

Research paper thumbnail of The Role of the Sarcomere and Cytoskeleton in Cardiac Mechanotransduction

The basic contractile unit of the cardiac myocyte is the sarcomere. Force develops as a result of... more The basic contractile unit of the cardiac myocyte is the sarcomere. Force develops as a result of the interaction of myosin heads with the actin thin filament. Actin filaments are directly connected to the Z line of the sarcomere, whereas myosin filaments are secured via the giant elastic protein titin. When cardiac muscle is stretched there is an immediate increase in contractility. This is an acute and fundamental cardiac adaptive response to an increase in demand. Evidence suggests that an increase in the probability of crossbridge formation, through titin strain and positive cooperative mechanisms, underlies the length-dependent activation of cardiac muscle. The sarcomere is connected to the sarcolemma by cytoskeletal components which link the Z-line with the membrane-spanning integrins and dystroglycan complex. Integrins and dystroglycan, in turn, bind to components of the extracellular matrix, such as laminin, which sheath the cardiac myocyte. Connections also exist between Z-line and nucleus via the intermediate filament protein desmin. The intracellular connections between the Z-line of the sarcomere and the sarcolemma allow transmission of force developed by the myofilaments. However, the physical pathway that links the extracellular matrix, membrane-spanning proteins, and the cell interior also plays a fundamental role in mechanotransduction. These links allow the cell to sense and respond to mechanical stimuli through connections with the cytoskeleton and activation of signalling cascades.