Novel method to alter length and load in isolated mammalian cardiac myocytes

1994 ◽  
Vol 267 (4) ◽  
pp. H1619-H1629 ◽  
Author(s):  
S. J. Sollott ◽  
E. G. Lakatta
Keyword(s):  
Ventricular Myocytes ◽  
Contractile Function ◽  
Single Cells ◽  
Elastic Tube ◽  
Cell Length ◽  
Fibrin Matrix ◽  
Time Integral ◽  
Myofilament Activation ◽  
Novel Method ◽  
Slack Length

We have devised a novel technique enabling reversible gradations in the resting and contraction length of intact mammalian ventricular myocytes of up to 15-18% over slack length. Enzymatically isolated single cells are embedded in a transparent, elastic, cross-linked fibrin matrix, contained within a narrow elastic tube. Reversible gradations in cell length are produced via fibrin matrix stretch, produced by stretching the tube. Simultaneous measurement of cell length, edge motion, and indo 1 fluorescence during auxotonic contractions permits characterization of cell contractile function. Although force cannot be directly measured, the time integral of contractile force (i.e., relative contractile impulse, a contractile index that is independent of shortening constraints) is derived combining myocyte shortening and matrix loading. Relatively small degrees of myocyte stretch produce a lightly afterloaded model dominated by variations in preload in which there is parallel augmentation of shortening and contractile impulse (force) development. At higher degrees of stretch, significant afterloading is introduced, resulting in the development of an inverse relationship between shortening and impulse (approaching isometric conditions). Length-dependent Ca2+ myofilament activation and load-dependent relaxation are readily demonstrated in intact isolated mammalian ventricular myocytes.

Morphological and contractile characteristics of rat cardiac myocytes from maturation to senescence

1989 ◽  
Vol 257 (1) ◽  
pp. H259-H265 ◽  
Author(s):  
A. Fraticelli ◽  
R. Josephson ◽  
R. Danziger ◽  
E. Lakatta ◽  
H. Spurgeon
Keyword(s):  
Time Course ◽  
Ventricular Myocytes ◽  
Cell Length ◽  
Maximal Velocity ◽  
Shortening Velocity ◽  
Average Cell ◽  
Time To Peak ◽  
Average Cell Volume ◽  
Slack Length ◽  
Contractile Characteristics

Morphological and contractile characteristics of individual myocytes isolated from rats of 2, 6–9, and 24–25 mo of age were measured. The average myocyte length measured under high power light microscopy in unattached cells increased from 133 microns at 2 mo to 146 microns at 6–9 mo to 162 microns at 24–25 mo of age. The average slack sarcomere length was 1.85, 1.83, and 1.82 microns at 2, 6–9, and 24–25 mo, respectively. The average cell volume measured via Coulter counter techniques approximately doubled between 2 and 24 mo. During the electrically stimulated twitch in individual unloaded myocytes contracting from slack length the absolute amplitude of cell shortening increased with age, but when expressed relative to cell length or as sarcomere shortening the age effect was obliterated. Neither the maximal velocity of myocyte shortening (cell length/s) nor the calculated maximal sarcomere shortening velocity varied with age. The time course of the twitch increased with aging, due largely to an increase in the time to peak shortening. Thus aging is associated with an increase in cell size due to the addition of sarcomeres. Except for a prolonged time course, the twitch contraction characteristics normalized for cell length and sarcomere number in unloaded ventricular myocytes contracting over a range of sarcomere lengths below the slack length do not change appreciably with age.

scholarly journals Enhanced activity of ventricular Na+-HCO3− cotransport in pressure overload hypertrophy

2007 ◽  
Vol 293 (2) ◽  
pp. H1254-H1264 ◽  
Author(s):  
Taku Yamamoto ◽  
Takeshi Shirayama ◽  
Tomohiko Sakatani ◽  
Tomosaburo Takahashi ◽  
Hideo Tanaka ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Single Cells ◽  
Ischemia Reperfusion ◽  
Pressure Overload ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Aortic Constriction ◽  
Ph Indicator ◽  
Adult Rat ◽  
Acid Extrusion

The Na+-HCO3− cotransporter (NBC) plays a key role in intracellular pH (pHi) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pHi was measured in single cells using the fluorescent pH indicator 2′,7′-bis(2-carboxyethyl)5-( 6 )carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC ( JNBC) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na+/H+ exchange was also increased in hypertrophied myocytes, the relative enhancement of JNBC was larger, 6) membrane depolarization markedly increased JNBC in hypertrophied myocytes, and 7) losartan, an ANG II AT1 receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na+ overload, which may render the ventricles more vulnerable to Ca2+ overload during ischemia-reperfusion.

scholarly journals Computational modeling of Takotsubo cardiomyopathy: effect of spatially varying β-adrenergic stimulation in the rat left ventricle

2014 ◽  
Vol 307 (10) ◽  
pp. H1487-H1496 ◽  
Author(s):  
Sander Land ◽  
Steven A. Niederer ◽  
William E. Louch ◽  
Åsmund T. Røe ◽  
Jan Magnus Aronsen ◽  
...  
Keyword(s):  
Left Ventricle ◽  
Takotsubo Cardiomyopathy ◽  
Troponin I ◽  
Contractile Function ◽  
Single Cells ◽  
Apical Ballooning ◽  
Calcium Sensitivity ◽  
Takotsubo Syndrome ◽  
Adrenergic Stimulation ◽  
Inotropic Effects

In Takotsubo cardiomyopathy, the left ventricle shows apical ballooning combined with basal hypercontractility. Both clinical observations in humans and recent experimental work on isolated rat ventricular myocytes suggest the dominant mechanisms of this syndrome are related to acute catecholamine overload. However, relating observed differences in single cells to the capacity of such alterations to result in the extreme changes in ventricular shape seen in Takotsubo syndrome is difficult. By using a computational model of the rat left ventricle, we investigate which mechanisms can give rise to the typical shape of the ventricle observed in this syndrome. Three potential dominant mechanisms related to effects of β-adrenergic stimulation were considered: apical-basal variation of calcium transients due to differences in L-type and sarco(endo)plasmic reticulum Ca2+-ATPase activation, apical-basal variation of calcium sensitivity due to differences in troponin I phosphorylation, and apical-basal variation in maximal active tension due to, e.g., the negative inotropic effects of p38 MAPK. Furthermore, we investigated the interaction of these spatial variations in the presence of a failing Frank-Starling mechanism. We conclude that a large portion of the apex needs to be affected by severe changes in calcium regulation or contractile function to result in apical ballooning, and smooth linear variation from apex to base is unlikely to result in the typical ventricular shape observed in this syndrome. A failing Frank-Starling mechanism significantly increases apical ballooning at end systole and may be an important additional factor underpinning Takotsubo syndrome.

Differential effects of anesthetics on in vivo skeletal muscle contractile function in the mouse

1996 ◽  
Vol 80 (1) ◽  
pp. 332-340 ◽  
Author(s):  
C. P. Ingalls ◽  
G. L. Warren ◽  
D. A. Lowe ◽  
D. B. Boorstein ◽  
R. B. Armstrong
Keyword(s):  
Skeletal Muscle ◽  
Contractile Function ◽  
Average Power ◽  
Eccentric Contraction ◽  
Peak Torque ◽  
High Dose ◽  
Skeletal Muscle Function ◽  
Contractile Responses ◽  
Time Integral

The purpose of this study was to evaluate the effects of four anesthetic regimens on in vivo contractile function of mouse ankle dorsiflexor muscles. The torque-frequency and torque-velocity relationships were determined for the following anesthetics: fentanyl-droperidol and diazepam (F-d/d); ketamine and xylazine (K/x); pentobarbital sodium (Ps); and methoxyflurane (Mf). Mf, Ps, and F-d/d regimens resulted in comparable contractile responses at low doses, whereas K/x produced a relative depression in isometric contractile function as shown by a decrease in the torque-time integral at the 300-Hz stimulation frequency (-13.9%; P < 0.05). Moreover, K/x caused a shift to the left in the torque-frequency curve as indicated by increases in torque-time integrals at 25 and 50 Hz. Both Ps and F-d/d regimens exhibited dose-dependent effects during the isovelocity contractions. Ps significantly reduced work (-28.7%) and average power (-28.9%) at 800 degrees/s at the high dose. In contrast, F-d/d anesthesia resulted in increases in peak torque (16-20%) and work (15-18%) output at all eccentric contraction velocities at the high dose, whereas average power was increased only at -800 (17%) and -1,000 degrees/s (17%). In conclusion, commonly used anesthetic regimens can affect the contractile response in vivo; K/x and Ps yield smaller torque outputs, whereas Mf and F-d/d consistently produce larger contractile responses. Mf and F-d/d are recommended for use in studying skeletal muscle function in mice in vivo.

Myocardial energetics during isometric twitch contractions of cat papillary muscle

1979 ◽  
Vol 236 (2) ◽  
pp. H244-H253 ◽  
Author(s):  
G. Cooper
Keyword(s):  
Linear Relationship ◽  
Isometric Tension ◽  
Active Tension ◽  
Continuous Increase ◽  
Time Integral ◽  
Myocardial Energetics ◽  
Isometric Twitch ◽  
Slack Length ◽  
Energy Dependent ◽  
Complete Contraction

During myocardial tetanus, when activation is maximum and constant, there is a linear relationship throughout contraction between oxygen consumption (MVo2) and the cumulative product of active tension and time (integral of AT). The goal of this study was to determine the relation of MVo2 to integral of AT during isometric myocardial twitch contractions. Ten right ventricular cat papillary muscles were studied in a flow respirometer. MVo2 was determined during contractions unloaded from Lmax to a slack length at successive 100-ms intervals after stimulation. In contrast to the linear relationship observed during tetanus, MVo2/integral of AT varied during twitch contractions: when the muscles were made slack 100 ms after stimulation MVo2/integral of AT was 389 +/- 51 (SE) (nl of O2/mg of dry muscle)/(N of active tension/mm2.s of active tension). This value was 94 +/- 7 at peak active tension and was constant thereafter. There was a continuous increase in cumulative MVo2 as integral of AT increased; before integral of AT began, MVo2 was 0.41 +/- 0.04 (nl of O2/mg)/contraction at Lmax and 0.22 +/- 0.04 at a slack length; at peak isometric tension MVo2 was 1.84 +/- 0.19; for a complete contraction MVo2 was 2.89 +/- 0.25. These data support two concepts 1) activation energy is small and dependent on initial length and tension; and 2) integral of AT is variably energy dependent throughout the entire isometric twitch contraction.

Growth in cell length in the fission yeast Schizosaccharomyces pombe

1985 ◽  
Vol 75 (1) ◽  
pp. 357-376 ◽  
Author(s):  
J.M. Mitchison ◽  
P. Nurse
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Size ◽  
Single Cells ◽  
Temperature Shift ◽  
Time Lapse ◽  
Cell Length ◽  
Wild Type ◽  
Cycle Growth ◽  
A Cell ◽  
Mutant Cells

The cylindrical cells of Schizosaccharomyces pombe grow in length by extension at the ends and not the middle. At the beginning of the cell cycle, growth is restricted to the ‘old end’, which existed in the previous cycle. Later on, the ‘new end’, formed from the septum, starts to grow at a point in the cycle that we have called NETO (‘new end take-off’). Fluorescence microscopy on cells stained with Calcofluor has been used to study NETO in size mutants, in blocked cdc mutants and with different growth temperatures and media. In wild-type cells (strain 972) NETO happens at 0.34 of the cycle with a cell length of 9.5 microns. With size mutants that are smaller at division, NETO takes place at the same size (9.0-9.5 microns) but this is not achieved until later in the cycle. Another control operates in larger size mutants since NETO occurs at the same stage of the cycle (about 0.32) as in wild type but at a larger cell size. This control is probably a requirement to have completed an event in early G2, since most cdc mutant cells blocked before this point in the cycle do not show NETO whereas most of those blocked in late G2 do show it. We conclude that NETO only happens if: (1) the cell length is greater than a critical value of 9.0-9.5 microns; and (2) the cell has traversed the first 0.3-0.35 of the cycle and passed early G2. NETO is delayed in poor media, in which cell size is also reduced. Temperature has little effect on NETO under steady-state conditions, but there is a transient delay for some hours after a temperature shift. NETO is later in another wild-type strain, 132. Time-lapse photomicrography was used to follow the rates of length growth in single cells. Wild-type cells showed two linear segments during the first 75% of the cycle. There was a rate-change point (RCP), coincident with NETO, where the rate of total length extension increased by 35%. This increase was not due simply to the start of new-end growth, since old-end growth slowed down in some cells at the RCP. cdc 11.123 is a mutant in which septation and division is blocked at 35 degrees C but nuclear division continues.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+ signals obtained with multiple indicators in mammalian vascular muscle cells

1987 ◽  
Vol 253 (6) ◽  
pp. H1456-H1461
Author(s):  
T. T. DeFeo ◽  
G. M. Briggs ◽  
K. G. Morgan
Keyword(s):  
Portal Vein ◽  
Calcium Concentration ◽  
Fluorescent Dyes ◽  
Contractile Function ◽  
Single Cells ◽  
Fluorescent Intensity ◽  
Fura 2 ◽  
Multiple Indicators ◽  
Calcium Storage ◽  
Vascular Muscle Cells

Enzymatically isolated single cells from ferret portal vein were loaded with the fluorescent dyes fura-2 and chlortetracycline. Ferret portal vein intact strips were loaded with the luminescent indicator aequorin. At short loading times, fura-2 loading resulted in relatively homogeneous images of labeled cells. At longer loading times, extremely heterogeneous images were obtained that were similar to those produced by chlortetracycline, an indicator recognized to enter calcium-storage organelles. A significant effect of fura-2 on contractile function was detected at the long but not at the short loading time. Caffeine, which is known to deplete calcium from sarcoplasmic reticulum, decreased the fura-2 fluorescent intensity when cells were incubated for a long loading time but caused no statistically significant change at the short loading time. Caffeine caused no drop in the aequorin signal but did cause a drop in the chlortetracycline fluorescence. These results are consistent with the idea that aequorin reports cytoplasmic intracellular calcium concentration ([Ca2+]i), chlortetracycline reports stored calcium, and fura-2 reports a mixed signal from the cytoplasm and calcium-storage organelles depending on incubation time.

Electrotonic suppression of early afterdepolarizations in isolated rabbit Purkinje myocytes

2000 ◽  
Vol 279 (1) ◽  
pp. H250-H259 ◽  
Author(s):  
Delilah J. Huelsing ◽  
Kenneth W. Spitzer ◽  
Andrew E. Pollard
Keyword(s):  
Ventricular Myocytes ◽  
Single Cells ◽  
Late Phase ◽  
Input Resistance ◽  
Membrane Resistance ◽  
Early Afterdepolarizations ◽  
Model Cell ◽  
Triggered Arrhythmias ◽  
Electrotonic Interactions ◽  
Coupling Current

Many studies suggest that early afterdepolarizations (EADs) arising from Purkinje fibers initiate triggered arrhythmias under pathological conditions. However, electrotonic interactions between Purkinje and ventricular myocytes may either facilitate or suppress EAD formation at the Purkinje-ventricular interface. To determine conditions that facilitated or suppressed EADs during Purkinje-ventricular interactions, we coupled single Purkinje myocytes and aggregates isolated from rabbit hearts to a passive model cell via an electronic circuit with junctional resistance ( R j). The model cell had input resistance ( R m,v) of 50 MΩ, capacitance of 39 pF, and a variable rest potential ( V rest,v). EADs were induced in Purkinje myocytes during superfusion with 1 μM isoproterenol. Coupling at high R j to normally polarized V rest,v established a repolarizing coupling current during all phases of the Purkinje action potential. This coupling current preferentially suppressed EADs in single cells with mean membrane resistance ( R m,p) of 297 MΩ, whereas EAD suppression in larger aggregates with mean R m,p of 80 MΩ required larger coupling currents. In contrast, coupling to elevated V rest,v established a depolarizing coupling current during late phase 2, phase 3, and phase 4 that facilitated EAD formation and induced spontaneous activity in single Purkinje myocytes and aggregates. These results have important implications for arrhythmogenesis in the infarcted heart when reduction of the ventricular mass due to scarring alters the R m,p-to- R m,v ratio and in the ischemic heart when injury currents are established during coupling between polarized Purkinje myocytes and depolarized ventricular myocytes.

Arachidonic acid enhances contraction and intracellular Ca2+ transients in individual rat ventricular myocytes

1997 ◽  
Vol 272 (1) ◽  
pp. H350-H359 ◽  
Author(s):  
D. S. Damron ◽  
B. A. Summers
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Muscle ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Receptor Activation ◽  
K Channels ◽  
Rat Ventricular Myocytes ◽  
Kinase C

Modulation of intracellular free Ca2+ concentration ([Ca2+]i) by inotropic stimuli alters contractility in cardiac muscle. Arachidonic acid (AA), a precursor for eicosanoid formation, is released in response to receptor activation and myocardial ischemia and has been demonstrated to alter K+ and Ca2+ channel activity. We investigated the effects of AA on contractility by simultaneously measuring [Ca2+]i and shortening in single field-stimulated rat ventricular myocytes. [Ca2+]i transients were measured using fura 2, and myocyte shortening was assessed using video edge detection. AA stimulated a doubling in the amplitude of the [Ca2+]i transient and a twofold increase in myocyte shortening. In addition, AA stimulated a 30% increase in the time to 50% diastolic [Ca2+]i and a 35% increase in the time to 50% relengthening. These effects of AA were mediated by AA itself (56 +/- 5%) and by cyclooxygenase metabolites. Pretreatment with the protein kinase C inhibitors staurosporine and chelerythrine nearly abolished (> 90% inhibition) these AA-induced effects. Inhibition of voltagegated K+ channels with 4-aminopyridine mimicked the effects of AA. Addition of AA to the 4-aminopyridine-treated myocyte had no additional effect on parameters of contractile function. These data indicate that AA alters the amplitude and duration of Ca2- transients and myocyte shortening via protein kinase C-dependent inhibition of voltage-gated K+ channels. Release of AA by phospholipases in response to receptor activation by endogenous mediators or pathological stimuli may be involved in mediating inotropic responses in cardiac muscle.

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