Ed White

The role of calcium in the response of cardiac muscle to stretch.

This review focuses on the complex interactions between two major regulators of cardiac function; Ca2+ and stretch. Initial consideration is given to the effect of stretch on myocardial contractility and details the rapid and slow increases in contractility. These are shown to be related to two diverse changes in Ca2+ handling (enhanced myofilament Ca2+ sensitivity and increased intracellular Ca2+ transient, respectively). Interaction between stretch and Ca2+ is also demonstrated with respect to the rhythm of cardiac contraction. Stretch has been shown to alter action potential configuration, generate stretch-activated arrhythmias, and increase the rate of beating of the sino-atrial node. A variety of Ca(2+)-dependent mechanisms including attenuation of Ca2+ extrusion via Na+/Ca2+ exchange, Ca2+ entry through stretch-activated channels (SACs) and mobilisation of intracellular Ca2+ stores have been proposed to account for the effect of stretch on rhythm. Finally, the interaction between stretch and Ca2+ in the secretion of natriuretic peptides and onset of hypertrophy is discussed. Evidence is presented that Ca2+ (entering through L-type Ca2+ channels or SACs, or released from sarcoplasmic reticular stores) influences secretion of both atrial and B-type natriuretic peptide; there is data to support both positive and negative modulation by Ca2+. Ca2+ also appears to be important in the pathway that leads to expression of precursors of hypertrophic protein synthesis. In conclusion, two of the major regulators of cardiac muscle function, Ca2+ and stretch, interact to produce effects on the heart; in general these effects appear to be additive.

The Frank–Starling mechanism in vertebrate cardiac myocytes

SUMMARY The Frank–Starling law of the heart applies to all classes of vertebrates. It describes how stretch of cardiac muscle, up to an optimum length, increases contractility thereby linking cardiac ejection to cardiac filling. The cellular mechanisms underlying the Frank–Starling response include an increase in myofilament sensitivity for Ca2+, decreased myofilament lattice spacing and increased thin filament cooperativity. Stretching of mammalian, amphibian and fish cardiac myocytes reveal that the functional peak of the sarcomere length (SL)–tension relationship occurs at longer SL in the non-mammalian classes. These findings correlate with in vivo cardiac function as non-mammalian vertebrates, such as fish, vary stroke volume to a relatively larger extent than mammals. Thus, it seems the length-dependent properties of individual myocytes are modified to accommodate differences in organ function, and the high extensibility of certain hearts is matched by the extensibility of their myocytes. Reasons for the differences between classes are still to be elucidated, however, the structure of mammalian ventricular myocytes, with larger widths and higher levels of passive stiffness than those from other vertebrate classes may be implicated.

Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte.

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 beta(2) adrenoceptor (AR) cAMP signal to the sarcolemmal compartment. Myocytes were treated with the cholesterol-depleting agent methyl-beta-cyclodextrin (M beta C) 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 beta(2) AR stimulation (with salbutamol in the presence of 1 microM 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 M beta C 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 beta(2) AR stimulation (P>0.05), but following caveolar disruption a 60-70% increase in phosphorylation of both proteins was seen, accompanied by positive lusitropy (P<0.05). These data show for the first time that disrupting caveolae converts the sarcolemmal-confined cAMP signal associated with beta(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 beta AR signalling in cardiovascular disease.

Do stretch-induced changes in intracellular calcium modify the electrical activity of cardiac muscle?

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. Myocardial stretch also modifies electrical activity and the opening of stretch-activated channels (SACs) is often used to explain this effect. However, the myocardium has many ionic currents that are regulated by [Ca(2+)](i) and in this review we discuss how stretch-induced changes in [Ca(2+)](i) can influence electrical activity via the modulation of these Ca(2+)-dependent currents. Our recent work in single ventricular myocytes has shown that axial stretch prolongs the action potential. This effect is sensitive to either SAC blockade by streptomycin or the buffering of [Ca(2+)](i) with BAPTA, suggesting that both SACs and [Ca(2+)](i) are important for the full effects of axial stretch on electrical activity to develop.

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

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 (Lmax). 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 [Ca2+]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.

Streptomycin and intracellular calcium modulate the response of single guinea-pig ventricular myocytes to axial stretch

We tested the hypothesis that both stretch‐activated channels (SACs) and intracellular calcium ([Ca2+]i) are important in the electrical response of single guinea‐pig ventricular myocytes to axial stretch. Myocytes were attached to carbon fibre transducers and stretched, sarcomere length increased by approximately 9 %, and there was a prolongation of the action potential duration. Streptomycin, a blocker of SACs, had no effect upon the shortening, [Ca2+]i transients or action potentials of electrically stimulated, unstretched myocytes, at a concentration of 50 μm, but at 40 μm, prevented any stretch‐induced increase in action potential duration. Under action potential clamp, stretch elicited a current with a linear current‐voltage relationship that was inward at membrane potentials negative to its reversal potential of −30 mV, in 10 of 24 cells tested, and was consistent with the activation of non‐specific, cationic SACs. This current was not seen in any stretched cells that were exposed to 40 μm streptomycin. However, exposure of cells to 5 μm BAPTA‐AM, in order to reduce [Ca2+]i transients, also abolished stretch‐induced prolongation of the action potential. We conclude that both SACs and [Ca2+]i are important in the electrical response of cardiac myocytes to stretch, and propose that stretch‐induced changes in electrical activity and [Ca2+]i may be linked by inter‐dependent mechanisms.

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

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. Methodology/Principal Findings Rat ventricular myocytes were exposed to solution of 0.02 tonicity (T; until lysis), 0.64T for 10–15 min (swelling), and/or methyl-β-cyclodextrin (MBCD; to disrupt caveolae). MBCD and 0.64T swelling reduced the number of caveolae visualised by electron microscopy by 75 and 50% respectively. MBCD stimulated translocation of caveolin 3 from caveolae-enriched buoyant membrane fractions, but both caveolin 1 and 3 remained in buoyant fractions after swelling. I Cl,swell inhibition in control cells decreased time to half-maximal volume (t 0.5,vol; 0.64T), consistent with a role for I Cl,swell in volume regulation. MBCD-treated cells showed reduced time to lysis (0.02T) and t 0.5,vol (0.64T) compared with controls. The negative inotropic response to swelling (an index of I Cl,swell activation) was enhanced by MBCD. Conclusions/Significance These data show that disrupting caveolae removes essential membrane reserves, which speeds swelling in hyposmotic conditions, and thereby promotes activation of I Cl,swell. They illustrate a general principle whereby caveolae as a membrane reserve limit increases in membrane tension during stretch/swelling thereby restricting mechanosensitive channel activation.

Arrhythmogenic substrate in hearts of rats with monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy

Mechanisms associated with right ventricular (RV) hypertension and arrhythmias are less understood than those in the left ventricle (LV). The aim of our study was to investigate whether and by what mechanisms a proarrhythmic substrate exists in a rat model of RV hypertension and hypertrophy. Rats were injected with monocrotaline (MCT; 60 mg/kg) to induce pulmonary artery hypertension or with saline (CON). Myocardial levels of mRNA for genes expressing ion channels were measured by real-time RT-PCR. Monophasic action potential duration (MAPD) was recorded in isolated Langendorff-perfused hearts. MAPD restitution was measured, and arrhythmias were induced by burst stimulation. Twenty-two to twenty-six days after treatment, MCT animals had RV hypertension, hypertrophy, and decreased ejection fractions compared with CON. A greater proportion of MCT hearts developed sustained ventricular tachycardias/fibrillation (0.83 MCT vs. 0.14 CON). MAPD was prolonged in RV and less so in the LV of MCT hearts. There were decreased levels of mRNA for K(+) channels. Restitution curves of MCT RV were steeper than CON RV or either LV. Dispersion of MAPD was greater in MCT hearts and was dependent on stimulation frequency. Computer simulations based on ion channel gene expression closely predicted experimental changes in MAPD and restitution. We have identified a proarrhythmic substrate in the hearts of MCT-treated rats. We conclude that steeper RV electrical restitution and rate-dependant RV-LV action potential duration dispersion may be contributing mechanisms and be implicated in the generation of arrhythmias associated with in RV hypertension and hypertrophy.

Visualization and quantification of whole rat heart laminar structure using high-spatial resolution contrast-enhanced MRI

It has been shown by histology that cardiac myocytes are organized into laminae and this structure is important in function, both influencing the spread of electrical activation and enabling myocardial thickening in systole by laminar sliding. We have carried out high-spatial resolution three-dimensional MRI of the ventricular myolaminae of the entire volume of the isolated rat heart after contrast perfusion [dimeglumine gadopentate (Gd-DTPA)]. Four ex vivo rat hearts were perfused with Gd-DTPA and fixative and high-spatial resolution MRI was performed on a 9.4T MRI system. After MRI, cryosectioning followed by histology was performed. Images from MRI and histology were aligned, described, and quantitatively compared. In the three-dimensional MR images we directly show the presence of laminae and demonstrate that these are highly branching and are absent from much of the subepicardium. We visualized these MRI volumes to demonstrate laminar architecture and quantitatively demonstrated that the structural features observed are similar to those imaged in histology. We showed qualitatively and quantitatively that laminar architecture is similar in the four hearts. MRI can be used to image the laminar architecture of ex vivo hearts in three dimensions, and the images produced are qualitatively and quantitatively comparable with histology. We have demonstrated in the rat that: 1) laminar architecture is consistent between hearts; 2) myolaminae are absent from much of the subepicardium; and 3) although localized orthotropy is present throughout the myocardium, tracked myolaminae are branching structures and do not have a discrete identity.

Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension

Pulmonary hypertension provokes right heart failure and arrhythmias. Better understanding of the mechanisms underlying these arrhythmias is needed to facilitate new therapeutic approaches for the hypertensive, failing right ventricle (RV). The aim of our study was to identify the mechanisms generating arrhythmias in a model of RV failure induced by pulmonary hypertension. Rats were injected with monocrotaline to induce either RV hypertrophy or failure or with saline (control). ECGs were measured in conscious, unrestrained animals by telemetry. In isolated hearts, electrical activity was measured by optical mapping and myofiber orientation by diffusion tensor-MRI. Sarcoplasmic reticular Ca(2+) handling was studied in single myocytes. Compared with control animals, the T-wave of the ECG was prolonged and in three of seven heart failure animals, prominent T-wave alternans occurred. Discordant action potential (AP) alternans occurred in isolated failing hearts and Ca(2+) transient alternans in failing myocytes. In failing hearts, AP duration and dispersion were increased; conduction velocity and AP restitution were steeper. The latter was intrinsic to failing single myocytes. Failing hearts had greater fiber angle disarray; this correlated with AP duration. Failing myocytes had reduced sarco(endo)plasmic reticular Ca(2+)-ATPase activity, increased sarcoplasmic reticular Ca(2+)-release fraction, and increased Ca(2+) spark leak. In hypertrophied hearts and myocytes, dysfunctional adaptation had begun, but alternans did not develop. We conclude that increased electrical and structural heterogeneity and dysfunctional sarcoplasmic reticular Ca(2+) handling increased the probability of alternans, a proarrhythmic predictor of sudden cardiac death. These mechanisms are potential therapeutic targets for the correction of arrhythmias in hypertensive, failing RVs.