Short‐term (6 weeks) voluntary wheel running exercise in young female rats that were in an active growth phase resulted in whole‐heart hypertrophy and myocyte concentric hypertrophy, when compared to sedentary controls. The cross‐sectional area of ventricular myocytes from trained rats was significantly greater than for those isolated from sedentary rats, with the greatest change in morphology seen in sub‐endocardial cells. There was no statistically significant effect of training on cell shortening in the absence of external mechanical loading, in [Ca2+]i transients, or in myofilament Ca2+ sensitivity (assessed during re‐lengthening following tetanic stimulation). Under the external mechanical load of carbon fibres, absolute force developed in myocytes from trained rats was significantly greater than in those from sedentary rats. This suggests that increased myocyte cross‐sectional area is a major contractile adaptation to exercise in this model. Training did not alter the passive mechanical properties of myocytes or the relative distribution of titin isomers, which was exclusively of the short, N2B form. However, training did increase the steepness of the active tension‐sarcomere length relationship, suggesting an exercise‐induced modulation of the Frank‐Starling mechanism. This effect would be expected to enhance cardiac contractility. Training lengthened the action potential duration of sub‐epicardial myocytes, reducing the transmural gradient in action potential duration. This observation may be important in understanding the cellular causes of T‐wave abnormalities found in the electrocardiograms of some athletes. Our study shows that voluntary exercise modulates the morphological, mechanical and electrical properties of cardiac myocytes, and that this modulation is dependent upon the regional origin of the myocytes.

Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a β-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.

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.

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.

. Visualization and quantification of whole rat heart laminar structure using highspatial resolution contrast-enhanced MRI. Am J Physiol Heart Circ Physiol 302: H287-H298, 2012. First published October 21, 2011; doi:10.1152/ajpheart.00824.2011.-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. myocardium; magnetic resonance imaging; ventricles; small animal imaging MYOCARDIAL STRUCTURE IS CENTRALLY important to cardiac mechanical function in health and disease (6), and myolaminar sliding is thought to be the principle mechanism of myocardial thickening in systole (10). Myolaminar structure has recently been shown to substantially influence the spread of activation in the myocardium (8,20).The myocardium is structured as stacked laminae of myocytes 4 -6 cells thick (ϳ80 -120 m), also known as myolaminae or sheets (24). These laminae are organized together in a complex fashion in which there are some regions of abrupt transmural change in laminar organization. The long axes of the myocytes, which make up the laminae, have a regular helical organization: the orientation of their long axes (with respect to the cardiac short axis) varies through ϳ120°trans-murally from endocardium to epicardium (33). The average orientation of the long axes of neighboring myocytes is known as the fiber-orientation (16). The myocardium therefore has regular lower order fiber architecture, with a more irregular and locally distinct higher order laminar arc...

BackgroundMany 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 FindingsRat 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/SignificanceThese 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.

Caveolae modulate ECC via the efficiency of the Ca(2+)-induced Ca(2+) release process, rather than Ca(2+) influx. Our data are also consistent with the hypothesis that interaction of G(i) protein cascade components with caveolin in the caveolae is necessary for effective signalling by this pathway. This suggests that changes in caveolin expression in the adult heart seen during aging and in disease will have consequences for baseline cardiac function and beta-adrenergic responsiveness.

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.