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Multiphoton microscopy is a powerful, non-invasive technique to image biological specimens. One current limitation of multiphoton microscopy is resolution as many of the biological molecules and structures investigated by research groups are similar in size or smaller than the diffraction limit. To date, the combination of multiphoton and super-resolution imaging has proved technically challenging for biology focused laboratories to implement. Here we validate that the commercial super-resolution Airyscan detector from ZEISS, which is based on image scanning microscopy, can be integrated under warranty with a pulsed multi-photon laser to enable multiphoton microscopy with super-resolution. We demonstrate its biological application in two different imaging modalities, second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), to measure the fibre thicknesses of collagen and elastin molecules surpassing the diffraction limit by a factor of 1.7±0.3x and 1.4±0.3x respectively, in human heart and lung tissues, and 3-dimensional in vitro models. We show that enhanced resolution and signal-to-noise of SHG using the Airyscan compared to traditional GaAs detectors allows for automated and precise measurement of collagen fibres using texture analysis in biological tissues., Competing Interests: The authors have declared that no competing interests exist.

Cardiac contractility increases as sarcomere length increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. The capacity of myosin to bind with actin and generate force in a muscle cell is Ca(2+) regulated by thin-filament proteins and spatially regulated by sarcomere length as thick-to-thin filament overlap varies. One mechanism underlying greater cardiac contractility as sarcomere length increases could involve longer myosin attachment time (ton) due to slowed myosin kinetics at longer sarcomere length. To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 μm sarcomere lengths. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate and MgATP binding rates. As MgATP increased, ton decreased for both sarcomere lengths, but ton was roughly 70% longer for 2.2 vs. 1.9 μm sarcomere length at maximally activated conditions. These ton differences were driven by a slower MgADP release rate at 2.2 μm sarcomere length (41 ± 3 vs. 74 ± 7 s(-1)), since MgATP binding rate was not different between the two sarcomere lengths. At submaximal activation levels near the pCa50 value of the tension-pCa relationship for each sarcomere length, length-dependent increases in ton were roughly 15% longer for 2.2 vs. 1.9 μm sarcomere length. These changes in cross-bridge kinetics could amplify cooperative cross-bridge contributions to force production and thin-filament activation at longer sarcomere length and suggest that length-dependent changes in myosin MgADP release rate may contribute to the Frank-Starling relationship in the heart., (Copyright © 2015 the American Physiological Society.)

Background: This study compared the influence of fasting/refeeding cycles and food restriction on rat myocardial performance and morphology., Methods: Sixty-day-old male Wistar rats were submitted to food ad libitum (C), 50% food restriction (R50), and fasting/refeeding cycles (RF) for 12 weeks. Myocardial function was evaluated under baseline conditions and after progressive increase in calcium and isoproterenol. Myocardium ultrastructure was examined in the papillary muscle., Results: Fasting/refeeding cycles maintained rat body weight and left ventricle weight between control and food-restricted rats. Under baseline conditions, the time to peak tension (TPT) was more prolonged in R50 than in RF and C rats. Furthermore, the maximum tension decline rate (-dT/dt) increased less in R50 than in RF with calcium elevation. While the R50 group showed focal changes in many muscle fibers, such as the disorganization or loss of myofilaments, polymorphic mitochondria with disrupted cristae, and irregular appearance or infolding of the plasma membrane, the RF rats displayed few alterations such as loss or disorganization of myofibrils., Conclusion: Food restriction promotes myocardial dysfunction, not observed in RF rats, and higher morphological damage than with fasting/refeeding. The increase in TPT may be attributed possibly to the disorganization and loss of myofibrils; however, the mechanisms responsible for the alteration in -dT/dt in R50 needs to be further clarified., (Copyright 2010 Elsevier Inc. All rights reserved.)

Although the myotendinous junction (MTJ) of skeletal striated muscle is well known, more detailed studies regarding the structure of the cardiac MTJ are scarce. The objective of the present study was to investigate the morphological characteristics of the MTJ in hearts of healthy and hypertensive (SHR) female rats using histological, ultrastructural and three-dimensional (SEM) methods, as well as to evaluate the expression of vinculin by immunofluorescence. In the two groups, light microscopy showed branching tendinous cords and collagen bundles penetrating the apex of the finger-like projections of the papillary muscle. SEM analysis revealed an enlarged apex of the papillary muscle in SHR which was not observed in healthy animals. The loss of force transmission appears to be compensated by the amplified connection between the papillary muscle and valvular collagen. A large number of intercalated disks close to the fiber apex, small amounts of an amorphous intercellular substance and numerous vesicles were observed in SHR. In these animals, the expression of vinculin was more marked showing a regular distribution and a pattern of transverse striations along the sarcolemma. The presence of this protein in transverse bands suggests that vinculin surrounds myofibrils in the region of the Z band. Vinculin staining was also more marked in the region of the tendinous cord-papillary muscle junctions of SHR compared to control animals. Vinculin was quantified by electrophoresis and higher amounts of this protein were observed in SHR compared to control animals.

Peculiarities of viscoelastic behavior of rabbit papillary muscle in passive state are studied by transversal versus longitudinal deformation curves, stress-strain and hysteresis curves, and stress relaxation curves under ramp stretching. The papillary muscle was chosen because of mostly longitudinal orientation of fibers and its elongated shape, which both make it as an appropriate model for uniaxial tests. The problem of evaluation of connective tissue protein structures and intracellular matrix contribution into the properties under consideration is solved by using the maceration method to remove intracellular structures. The different contribution of intracellular and extracellular protein features into total properties of a papillary muscle leads to nonlinearity of myocardial viscoelastic properties, such as the increase of differential elastic module and relaxation time with deformation.

Background: The effects of aging on papillary muscle have been widely demonstrated, but no data on the effects of exercise on the age-related changes are available., Objective: To analyze the effects of aging on the morphological and quantitative properties of papillary muscle and investigate whether a long-term moderate exercise program would exert a protective effect against the effects of aging., Methods: We used electron microscopy to study the density of myocytes, capillaries and connective tissue and the cross-sectional area of myocytes of the papillary muscle of the left ventricle of 6- and 13-month-old untrained and exercised Wistar rats., Results: As expected, the volume density of myocytes declined significantly (p<0.05) with aging. The length density of myocardial capillaries also declined with aging, but not significantly. The interstitial volume fraction of the papillary muscle tissue increased significantly (P<0.05) with age. The number of myocyte profiles showed a reduction of 20% that was accompanied by myocyte hypertrophy in the aged rats (P<0.05). Animals submitted to a 60-minute daily session,, 5 days/wk at 1.8 km x h(-1) of moderate running on a treadmill for 28 weeks showed a reversion of all the observed aging effects on papillary muscle., Conclusion: The present study supports the concept that long-term exercise training restrains the aging-related deleterious changes in the papillary muscle.

Background: Cardiotin expression is observed in adult cardiac tissue. In the present study, we provide evidence for the specific localization of cardiotin in cardiac mitochondria and for its down-regulation during adaptive remodeling (dedifferentiation) of cardiomyocytes., Methods: Immunocytochemistry was used to study cardiotin localization in adult rabbit papillary muscle, in late-stage embryonic rabbit left ventricular tissue, and in left ventricle samples of rabbits suffering from pressure and volume overload. Western blot analysis of cardiotin was performed in purified pig heart mitochondrial fractions. Cardiotin expression was monitored in vitro in isolated adult rat and rabbit left ventricular cardiomyocytes., Results: Western blot analysis revealed the presence of cardiotin in the mitochondrial fractions of pig heart. Immunoelectron microscopy confirmed the presence of cardiotin in cardiac mitochondria of normal adult rabbits both in vivo and in vitro. Quantification of the localization of immunogold particles suggests an association of cardiotin with the mitochondrial inner membrane. Cardiotin expression is initiated in late-stage embryonic rabbit heart, whereas in adult ventricular tissue cardiotin clearly stained longitudinal arrays of mitochondria. Pressure- and volume-overloaded myocardium showed a reduction in cardiotin expression in dispersed local myocardial areas. Cell cultures of adult cardiomyocytes showed a gradual loss in cardiotin expression in parallel with a sarcomeric remodeling., Conclusions: Our results demonstrate the specific localization of cardiotin in adult cardiomyocyte mitochondria and propose its use as an early marker for cardiomyocyte adaptive remodeling and dedifferentiation.

Aims: An intriguing difference between vertebrate skeletal and cardiac muscles is that the lengths of the thin filaments are constant in the former but variable in the latter. The thick filaments have constant lengths in both types of muscles. The contractile behaviour of a muscle is affected by the lengths of both types of filaments as the tension generated during contraction depends on the amount of filament overlap. To understand the behaviour of cardiac muscle, it is important to know the distribution of the thin filament lengths. The previous detailed analysis by Robinson and Winegrad used serial transverse sections to determine the lengths of the thin filaments. However, the precision, set by the 100 nm section thickness, was low. Here, we have used electron tomography to produce 3D images of rat and mouse cardiac muscles in which we can actually see individual thin filaments up to the free ends and see that these free ends have variable locations. For comparison, we also measure the thin filament lengths in skeletal muscle (frog sartorius)., Methods and Results: Cardiac papillary muscles were obtained from a rat (Sprague-Dawley) and a mouse (C57/B6). Skeletal muscle (sartorius) was obtained from a frog (Rana pipiens). Longitudinal sections (100 nm thick) were used to produce tilt series and tomograms from which the thin filament paths were traced. Cardiac papillary muscle thin filaments in rat and mouse range from 0.94 to 1.10 microm, with a mean length of 1.04 microm and standard deviation of 0.03 microm. For frog sartorius muscle, the thin filament length was 0.94 microm with standard deviation of 0.01 microm., Conclusion: Electron tomography of cardiac and skeletal muscles allows direct visualization and high precision measurement of the lengths of thin filaments.

This study evaluated the effects of exercise training on myocardial function and ultrastructure of rats submitted to different levels of food restriction (FR). Male Wistar-Kyoto rats, 60 days old, were submitted to free access to food, light FR (20%), severe FR (50%) and/or to swimming training (one hour per day with 5% of load, five days per week for 90 days). Myocardial function was evaluated by left ventricular papillary muscle under basal condition (calcium 1.25 mM), and after extracellular calcium elevation to 5.2 mM and isoproterenol (1 microM) addition. The ultrastructure of the myocardium was examined in the papillary muscle. The training effectiveness was verified by improvement of myocardial metabolic enzyme activities. Both 20% and 50% food restriction protocols presented minor body and ventricular weights gain. The 20%-FR, in sedentary or trained rats, did not alter myocardial function or ultrastructure. The 50%-FR, in sedentary rats, caused myocardial dysfunction under basal condition, decreased response to inotropic stimulation, and promoted myocardial ultrastructural damage. The 50%-FR, in exercised rats, increased myocardial dysfunction under basal condition but increased response to inotropic stimulation although there was myocardial ultrastructural damage. In conclusion, the exercise training in severe restriction caused marked myocardial dysfunction at basal condition but increased myocardial response to inotropic stimulation.

The role of cardiac myosin binding protein-C (MyBP-C) on myocardial stiffness was examined in skinned papillary muscles of wild-type (WT(+/+)) and homozygous truncated cardiac MyBP-C (MyBP-C(t/t) male mice. No MyBP-C was detected by gel electrophoresis or by Western blots in the MyBP-C(t/t) myocardium. Rigor-bridge dependent myofilament stiffness, i.e., rigor minus relaxed stiffness, in the MyBP-C(t/t) myocardium (281 +/- 44 kN/m2) was 44% that in WT(+/+) (633 +/- 141 kN/m2). The center-to-center spacing between thick filaments as determined by X-ray diffraction in MyBP-C(t/t) (45.0 +/- 1.2 nm) was not significantly different from that in WT(+/+) (43.2 +/- 0.9 nm). The fraction of cross-sectional area comprised of myofibrils, as determined by electron microscopy, was reduced in the MyBP-C(t/t) (39.9%) by 10% compared to WT(+/+) (44.5%). These data suggest that the 56% reduction in rigor-bridge dependent stiffness of the skinned MyBP-C(t/t) myocardium could not be due solely to a 10% reduction in the number of thick filaments per cross-sectional area and must also be due to approximately 50% reduction in the stiffness of the rigor-bridge attached thick filaments lacking MyBP-C.

Background: Synchrotron radiation has been used to analyze crossbridge dynamics in isolated papillary muscle and excised perfused hearts with the use of x-ray diffraction techniques. We showed that these techniques can detect regional changes in rat left ventricle contractility and myosin lattice spacing in in situ ejecting hearts in real time. Furthermore, we examined the sensitivity of these indexes to regional ischemia., Methods and Results: The left ventricular free wall of spontaneously beating rat hearts (heart rate, 290 to 404 bpm) was directly exposed to brief high-flux, low-emittance x-ray beams provided at SPring-8. Myosin mass transfer to actin filaments was determined as the decrease in reflection intensity ratio (intensity of 1,0 plane over the 1,1 plane) between end-diastole and end-systole. The distance between 1,0 reflections was converted to a lattice spacing between myosin filaments. We found that mass transfer (mean, 1.71+/-0.09 SEM, n=13 hearts) preceded significant increases in lattice spacing (2 to 5 nm) during systole in nonischemic pericardium. Left coronary occlusion eliminated increases in lattice spacing and severely reduced mass transfer (P<0.01) in the ischemic region., Conclusions: Our results suggest that x-ray diffraction techniques permit real-time in situ analysis of regional crossbridge dynamics at molecular and fiber levels that might also facilitate investigations of ventricular output regulation by the Frank-Starling mechanism.

We examined the sarcomere length-dependence of the spacing of the hexagonal lattice of the myofilaments and the mass transfer of myosin cross-bridges during contraction of right ventricular papillary muscle of the rat. The lattice spacing and mass transfer were measured by using X-ray diffraction, and the sarcomere length was monitored by laser diffraction at the same time. Although the lattice spacing and the sarcomere length were inversely related, their relationship was not exactly isovolumic. The cell volume decreased by about 15% when the sarcomere length was shortened from 2.3 micro m to 1.8 micro m. Twitch tension increased with sarcomere length (the Frank-Starling law). At the peak tension, the ratio of the intensity of the (1,0) equatorial reflection to that of the (1,1) reflection was smaller when the tension was greater, showing that the larger tension at a longer sarcomere length accompanies a larger amount of mass transfer of cross-bridges from the thick to the thin filament. The result suggests that the Frank-Starling law is due to an increase in the number of myosin heads attached to actin, not in the average force produced by each head.

Collagen degradation is suggested to be responsible for long-term contractile dysfunction in different cardiomyopathies, but the effects of acute and specific collagen type I removal (main type in the heart muscle) on tension have not been studied. We determined the diastolic and developed tension length relations in isometric contracting perfused rat papillary muscles (perfusion pressure 60 cmH(2)O) before and after acute and specific removal of small collagen struts with the use of purified collagenase type I. At 95% of the maximal length (95%L(max)), diastolic tension increased 20.4 +/- 8.1% (P < 0.05, n = 6) and developed tension increased 15.0 +/- 6.7% after collagenase treatment compared with time controls. Treatment increased the diastolic muscle diameter by 7.1 +/- 3.4% at 95%L(max), whereas the change in diameter due to contraction was not changed. Diastolic coronary flow and normalized coronary arterial flow impediment did not change after collagenase treatment. Electron microscopy revealed that the number of small collagen struts, interconnecting myocytes, and capillaries was reduced to approximately 32% after treatment. We conclude that removal of the small collagen struts by acute and specific collagen type I degradation increases diastolic and developed tension in perfused papillary muscle. We suggest that diastolic tension is increased due to edema, whereas developed tension is increased because the removal of the struts poses a lower lateral load on the cardiac myocytes, allowing more myocyte thickening.

Spectrin and its associated proteins are essential for the integrity of muscle cells and there is increasing evidence for their involvement in signalling pathways as well as having a structural function in mediating stress. Spectrin is a multigene family and it is essential to determine which isoforms are present and their location in the cell. In heart muscle, we have found that one spectrin isoform, alphaII-spectrin, is strongly represented and, using immunofluorescence, we show that it lies within the contractile fibres near the Z-disc as well as on the cardiomyocyte plasma membrane. Electron microscopy of immunogold-labelled cryosections reveals statistically significant clustering of gold particles near the Z-disc, within and close to the edge of myofibrils. betaII-spectrin and ankyrin-R and G are both known to occupy this region. We suggest that alphaIIbetaII spectrin tetramers with ankyrin organise and/or stabilise cardiac muscle cell membrane components relative to the contractile apparatus.

Various mechanisms have been suggested to explain cardiac force-length Ca2+ relations. The existence of a cooperativity mechanism, whereby cross-bridge (XB) recruitment is affected by the number of active XBs, suggests that the force response to length oscillations should lag length oscillations. Consequently, the oscillatory force response should be larger during shortening than during lengthening. To test this prediction, force responses to large-sarcomere length (SL) oscillations (36.7 +/- 16.0 nm) at different SLs (n = 6) and frequencies (n = 7) were studied in intact tetanized trabeculae dissected from rat right ventricle (n = 13). Stable tetani were obtained by utilizing 30 microM cyclopiazonic acid in Krebs-Henseleit solution containing 6 mM extracellular Ca(2+) at 25 degrees C. SL was measured by laser diffraction techniques (Dalsa). Force was measured by silicone strain gauge. Instantaneous dynamic stiffness during large oscillations was measured by superimposing additional fast (50 or 200 Hz) and small-amplitude (2.25 +/- 0.25 nm) oscillations. The force responses lagged the SL oscillations at slow frequencies (112 +/- 41 ms at 1 Hz), and counterclockwise hystereses were obtained in the force-length plane: the force was higher during shortening than during lengthening. The delay in the force response decreased as the frequency of the SL oscillation was increased. Clockwise hysteresis, where the force preceded the SL, was obtained at frequencies >4 Hz. Similar hysteresis characteristics were obtained in the force-SL and stiffness-SL planes. Maximal lag was observed at the shortest SL, and the delay decreased with sarcomere elongation: 131.1 +/- 31.7 ms at 1.78 +/- 0.03 microm vs. 14.7 +/- 18.5 ms at 1.99 +/- 0.015 microm. The results establish the ability of cardiac fiber to adapt XB recruitment to changes in prevailing loading conditions. This study supports the stipulated existence of a cooperativity mechanism that regulates XB recruitment and highlights an additional method to characterize regulation of the force-length relation.

Cryoelectron microscopy makes it possible to record high-resolution detail from large and complex structures. However, its application to understanding cellular structure is limited by the requirement that samples should be no thicker than approximately 0.5-1 microm. Therefore it is important to develop the ability to section biological material so that it can be imaged in its native frozen state. Here we have adapted standard methods of preparing cryosections so that they can be imaged by cryoelectron microscopy. As used for immunolabeling, cryosections of chemically fixed, cryoprotected frozen rat cardiac muscle were thawed, applied to carbon-coated grids, and rinsed on a drop of buffer. The special step here is that the cryosections were then refrozen by being plunged into liquid ethane and imaged at approximately -180 degrees C in a 200-kV field-emission gun electron microscope. The unstained cryosections have good contrast, allowing the identification of optimum regions of the sample. Considerable fine detail is observed within the substructure of the sarcomere A-band and I-band. Fourier transform analysis of the micrographs shows that this method preserves high structural order, hence these sections are well-suited to 3D reconstruction. We conclude that this approach has considerable potential for obtaining intermediate- and high-resolution structural detail from bulk tissue.

Objective: The primary aim of this study was to investigate whether activation of Na influx via voltage-gated Na channels can elevate sub-sarcolemmal ('fuzzy-space') [Na] and transiently activate Na/K pump current (I(p))., Methods and Results: Initially, Na/K pump activity was characterised in whole-cell voltage-clamped single guinea-pig ventricular myocytes. I(p) was activated by intracellular Na with a K(m) of 15.5 mM and a Hill coefficient of 1.7. Extracellular K activated I(p) with a K(m) of 1.6 mM. In these experiments, a finite ouabain-sensitive I(p) was measured when the pipette [Na] was zero. This suggests that there is an accumulation of Na in a sub-sarcolemmal space that is not in equilibrium with the bulk cytosol (which is assumed to be efficiently dialysed by the low-resistance patch-pipettes used). Such a sub-sarcolemmal Na gradient was observed in separate experiments in intact rabbit papillary muscles using electron probe X-ray microanalysis. In these studies, a fuzzy-space of limited Na diffusion was observed 100-200 nm below the sarcolemmal membrane. This sub-sarcolemmal Na gradient was similar whether muscles were frozen at peak-systole or end-diastole suggesting that the fuzzy-space Na does not change over the course of the contractile cycle. This was further investigated in isolated guinea pig myocytes where evidence for a transient activation of I(p) was sought immediately after the activation of voltage-gated Na channels. A single clamp step from -80 to 0 mV activated Na influx but, in the 10-2000 ms immediately following the initial Na influx no evidence for a transient activation of I(p) was observed. Similarly, no activation of I(p) could be detected immediately following a train of 20 rapid (5-Hz) pulses designed to maximise Na influx., Conclusions: These studies provide evidence for the existence of a maintained sub-sarcolemmal elevation of [Na] in ventricular myocardium; however, this fuzzy-space [Na] did not change immediately after the activation of Na influx via voltage-gated Na channels or throughout the contractile cycle.

Transesophageal echocardiography was performed to evaluate the exact cause of severe mitral regurgitation in a 64-year-old man presented with hypotension and dyspnea after acute inferior wall myocardial infarction. In mid-esophageal two- and four-chamber view, the ruptured stump of papillary muscle could not be visualized. However, in transgastric two-chamber view, we could clearly visualize the ruptured head of the posteromedial papillary muscle as a separated mass attached by chorda tendinae, as well as the freely mobile stump of the ruptured papillary muscle within the left ventricle. So, the comprehensive transesophageal echocardiography, including transgastric imaging, is always indicated in patients with severe mitral regurgitation after acute myocardial infarction.

The relations between stress, stimulation rate and sarcomere length (SL) were investigated in 24 cardiac trabeculae isolated from right ventricles of mice (CF-1 males, 25-30 g) and superfused with Hepes solution ([Ca(2+)](o) = 1 mM, pH 7.4, 25 degrees C). Stress and SL were measured by a strain gauge transducer and laser diffraction technique, respectively. Stress versus stimulation frequency formed a biphasic relation (25 degrees C, [Ca(2+)](o) = 2 mM) with a minimum at 0.7-1 Hz (~15 mN mm(-2)), a 150 % decrease from 0.1 to 1 Hz (descending limb) and a 75 % increase from 1 to 5 Hz (ascending limb). Ryanodine (0.1 microM) inhibited specifically the descending limb, while nifedipine (0.1 microM) affected specifically the ascending limb. This result suggests two separate sources of Ca(2+) for stress development: (1) net Ca(2+) influx during action potentials (AP); and (2) Ca(2+) entry into the cytosol from the extracellular space during diastolic intervals; Ca(2+) from both (1) and (2) is sequestered by the SR between beats. Raising the temperature to 37 degrees C lowered the stress-frequency relation (SFR) by approximately 0-15 mN mm(-2) at each frequency. Because the amount of Ca(2+) carried by I(Ca,L) showed a approximately 3-fold increase under the same conditions, we conclude that reduced Ca(2+) loading of the SR was probably responsible for this temperature effect. A simple model of Ca(2+) fluxes addressed the mechanisms underlying the SFR. Simulation of the effect of inorganic phosphates (P(i)) on force production was incorporated into the model. The results suggested that O(2) diffusion limits force production at stimulation rates >3 Hz. The stress-SL relations from slack length (approximately 1.75 microm) to 2.25 microm showed that the passive stress-SL curve of mouse cardiac trabeculae is exponential with a steep increase at SL >2.1 microm. Active stress (at 1 Hz) increased with SL, following a curved relation with convexity toward the abscissa at [Ca(2+)] = 2 mM. At [Ca(2+)] from 4 to 12 mM, the stress-SL curves superimposed and the relation became linear, which revealed a saturation step in the activation of force production. EC coupling in mouse cardiac muscle is similar to that observed previously in the rat, although important differences exist in the Ca(2+) dependence of force development. These results may suggest a lower capacity of the SR for buffering Ca(2+), which makes the generation of force in mouse cardiac ventricle more dependent on Ca(2+) entering during action potentials, particularly at high heart rate.

Electron probe microanalysis was employed to determine the elemental concentration (K,Na,Cl) in a myocyte on cryosections of the papillary muscle of the isolated rat (Wistar) heart. Protocols of global ischemia and ischemic conditions under glucose-free anoxic perfusion were applied. It was shown that global ischemia induces potassium deficiency (94 +/- 2 mM) in the myocyte and an increase in the level of sodium (72 +/- 4 mM) and chlorine (42 +/- 1 mM) in the cytoplasm compared with intact cell (122 +/- 2; 36 +/- 1; 24 +/- 1 mM). Glucose-free anoxic perfusion leads to a smooth fall of potassium concentration in the cell up to 54 +/- 2 mM with the retention of intracellular sodium (40 +/- 1 mM) and chlorine (26 +/- 1 mM) level. The present finding suggest that, in early ischemia, specific membrane mechanisms of ion transport are activated. Among these are KNa channel, Hi(+)-Nao+ exchange, KATP channel, lactate transport from the cell, associated either with potassium efflux to the extracellular space or chlorine influx into the myocyte. It is assumed that Na/K-ATPase is also activated under ischemic conditions.

To determine whether and to what extent one component of the extracellular matrix, fibrillar collagen, contributes causally to abnormalities in viscoelasticity, collagen was acutely degraded by activation of endogenous matrix metalloproteinases (MMPs) with the serine protease plasmin. Papillary muscles were isolated from normal cats and cats with right ventricular pressure overload hypertrophy (POH) induced by pulmonary artery banding. Plasmin treatment caused MMP activation, collagen degradation, decreased the elastic stiffness constant, and decreased the viscosity constant in both normal and POH muscles. Thus, whereas many mechanisms may contribute to the abnormalities in myocardial viscoelasticity in the POH myocardium, changes in fibrillar collagen appear to play a predominant role.

Scanning electron microscopy was used to examine the chordae tendineae in young (approximately 20 years old) and old (approximately 85 years old) cadavers. A network of collagen fibrils was observed in both the outer and intersurface layers of the chordae tendineae. In young subjects, a regularly arranged series of disconnected collagenous ring-like structures surrounded the longitudinal collagen bundles forming the inner core of the chordae tendineae. In old subjects, the ring-like structures of the chordae tendineae were few or absent. The ring-like structures may be related to reducing mechanical stress during the tightening, twisting, and slackening of the chordae tendineae., (Copyright 2002 Wiley-Liss, Inc.)

Semi-immobilization of a partial area of the ventral edge, lateral epicardium of the left auricle (ventrolateral of left auricle), by using quick adhesion glue induces moderate hypertrophy of myocytes with an average increase of 34% in cross-sectional area. Intercellular connective tissues increased, and cellular sizes varied markedly. The ultrastructure of immobilized (semi-immobilized) myocytes commonly exhibited degenerating features in myofibrils, various cytoplasmic organelles including mitochondrial cristae and sarcoplasmic reticulum (SR) were disrupted, and T-tubules disappeared. Z-line streaming and widening (hypertrophic Z-line, rod bodies) and increase of metabolic particle deposition are typical phenomena in addition to intercalated disc (Id) disorganization. The results suggest that semi-immobilization of the auricle induces hypertrophy of myocytes in association with degeneration and disruption of myofibrils and other cytoplasmic organelles, and an increase of intercellular connective tissues, rather than increase of myofibril mass. This is the first study to immobilize only a part of the heart rather than the whole animal. Our results using artificial immobilization of cardiac myocytes were extremely significant since the structural alterations obtained were similar to that observed in cardiomyopathies. This suggests that myocytes progressing to heart failure are also subjected to inhibition of movement. Therefore, this experiment may prove very useful as a model for studying the functional effect of heart failure observed in cardiomyopathy.

Because the amount and structure of type I collagen are thought to affect the mechanics of ventricular myocardium, we investigated myocardial collagen structure and passive mechanical function in the osteogenesis imperfecta murine (oim) model of pro-alpha2(I) collagen deficiency, previously shown to have less collagen and impaired biomechanics in tendon and bone. Compared with wild-type littermates, homozygous oim hearts exhibited 35% lower collagen area fraction (P:<0.05), 38% lower collagen fiber number density (P:<0.05), and 42% smaller collagen fiber diameter (P:<0.05). Compared with wild-type, oim left ventricular (LV) collagen concentration was 45% lower (P:<0.0001) and nonreducible pyridinoline cross-link concentration was 22% higher (P:<0.03). Mean LV volume during passive inflation from 0 to 30 mm Hg in isolated hearts was 1.4-fold larger for oim than wild-type (P:=NS). Uniaxial stress-strain relations in resting right ventricular papillary muscles exhibited 60% greater strains (P:<0.01), 90% higher compliance (P:=0.05), and 64% higher nonlinearity (P:<0.05) in oim. Mean opening angle, after relief of residual stresses in resting LV myocardium, was 121+/-9 degrees in oim compared with 45+/-4 degrees in wild-type (P:<0.0001). Mean myofiber angle in oim was 23+/-8 degrees greater than wild-type (P:<0.02). Decreased myocardial collagen diameter and amount in oim is associated with significantly decreased fiber and chamber stiffness despite modestly increased collagen cross-linking. Altered myofiber angles and residual stress may be beneficial adaptations to these mechanical alterations to maintain uniformity of transmural fiber strain. In addition to supporting and organizing myocytes, myocardial collagen contributes directly to ventricular stiffness at high and low loads and can influence stress-free state and myofiber architecture.

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/- mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/- mice was due almost entirely to a reduction in the number of individual gap junctions (226 +/- 52 vs. 150 +/- 32 individual gap junctions/field in Cx43 +/+ and +/- ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.

Endothelin-1 (ET-1) induces severe pathologic conditions such as coronary spasm followed by vasospastic angina pectoris and acute myocardial infarction. The related pathophysiologic mechanisms have remained obscure. Endothelin-1 receptor (ET(A) and ET(B)) is reported to couple with several types of G protein-involved pathways that participate in phospholipase C activation and atrial myofibrils organization into sarcomeric units. Here we demonstrate that ET-1 induces histologic and pathologic dysfunction in the rabbit myocardium and that such pathologic events are prevented by the Rho-kinase inhibitor fasudil. Although the bolus injection of ET-1 (1.4 nmol/kg) via the auricular vein of the rabbit induced only transient T-wave elevation, irreversible, severe histologic changes were observed in papillary muscles of the ventricle, and multifocal myocardial necrosis with infiltration of neutrophils and macrophages in the left ventricle occurred. Oral administration of fasudil (10 mg/kg) significantly reduced the occurrence of myocardial injury determinants, whereas conventional Ca2+ channel blockers (nifedipine, diltiazem) and a K+ channel opener (nicorandil; 10 mg/kg, p.o. each) showed a lesser or no effect on such determinants. These results suggest that ET-1 induces severe myocardial dysfunction based not only on the occurrence of vasospastic ischemia but also on its direct effects on the myocardium.

Contractile dysfunctions have been demonstrated in different experimental models of diabetes which have similar characteristics to many of the abnormalities found in the clinical setting. Administration of streptozotocin (STZ) to young adult rats induces beta-cell necrosis of the pancreas which gives rise to hypoinsulinaemia and hyperglycaemia, features which are also seen in untreated type 1 clinical diabetes. We have investigated the chronic effects of STZ-induced diabetes on contraction in rat ventricular myocytes and ultrastructure of cardiac muscle. Diabetes was induced in male Wistar rats (230-270 g) with a single injection of STZ (60 mg kg(-1)). At 2 and 10 months after STZ treatment, the amplitude of contraction was larger in diabetic compared to control myocytes. Time to peak contraction was significantly longer at 2 months but appeared to normalise at 10 months after STZ treatment. In contrast, time to half relaxation of contraction was not significantly different after 2 months but was significantly reduced at 10 months after STZ treatment compared to control. Transmission electron microscope examination of cardiac muscle showed that the ultrastructure of cardiac muscle, especially structures associated with contraction, were not greatly altered after STZ treatment. Sarcomere lengths were not significantly different in papillary or ventricular muscle at 4 or 8 months after STZ treatment compared to control. Our data provide evidence that morphological defects in contractile myofilaments and associated structures cannot explain contractile dysfunctions seen in ventricular myocytes from STZ-treated animals.

The Ca(2+)-releasing mechanisms of the sarcoplasmic reticulum responsible for cardiac muscle contraction in carp were examined and compared with these mechanisms in rats. Morphologically, the ventricular muscles of the carp heart are composed of an outer compact and an inner spongy layer. In the present study, ventricular muscle preparations were obtained from the compact layer of the carp heart, because the spongy layer does not contribute significantly to the overall force of contraction. Electron microscopic observations showed that the sarcoplasmic reticulum in the carp ventricular muscle, compared to that in the rat ventricular muscle, was poorly developed. Consistent with this finding, specific [3H]ryanodine binding to partially purified sarcoplasmic reticulum preparations obtained from carp ventricular muscle as compared with the preparations isolated from the rat ventricular muscle showed a lower affinity and a smaller number of binding sites. Additionally, a higher Ca2+ concentration was required to cause a half maximal stimulation of [3H]ryanodine binding in the carp heart. In skinned ventricular muscle fibers isolated from carp hearts, the caffeine-induced contracture was significantly weaker than that observed in rat hearts. These results suggest that, in carp hearts, the sarcoplasmic reticulum has an important role as a supply source of Ca2+ for muscle contraction, though the storage capacity and/or amount of Ca2+ release in carp was significantly smaller than that in rats.

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.

The papillary muscles (PM) of the heart have been the subject of numerous structural and functional studies. However, despite the importance of the collagenous compartment of the heart in the mechanical and electrical properties of the myocardium, little information is available on the structural organization of collagen within the PM. We study here the structural organization of collagen within the mitral papillary muscles (PM) of the human heart. Fragments of human mitral PM from normal and hypertensive subjects were macerated in NaOH to eliminate the cellular components. Macerated and nonmacerated samples were then studied with the scanning electron microscope (SEM). SEM shows that cardiac myocytes and endomysial capillaries are ensheathed in a layer of collagenous tissue. The myocyte sheath wall is formed by thin collagen fibers oriented at right angles to the main cell axis. These sheaths are open structures, collagen fibers continuing into adjacent sheaths at the points of lateral communications. Thick perimysial septa do not divide the PM tissue into separate compartments. Hypertensive hearts show perivascular and interstitial fibrosis. In addition, the lumen of the coronary vessels is reduced or obliterated, and large areas of the myocardium are substituted by densely packed collagen. Endomysial sheaths constitute a continuous collagenous layer that replicates the myocyte network. The endomysium should play a complex role in myocardial mechanics, assuring the equal distribution of force during the cardiac cycle. The absence of insulating boundaries should facilitate lateral propagation of excitation. Fibrosis in hypertensive hearts appears to be both reactive and reparative. The increase in the amount of collagen should greatly impair contractile capabilities and electrical conductance, severely compromise heart function, and contribute to development of heart failure.

Objectives: To understand papillary muscle malformations, such as in parachute mitral valves or parachute-like asymmetric mitral valves, we studied the development of papillary muscles., Methods: Normal human hearts at between 5 and 19 weeks of development were studied with immunohistochemistry, three-dimensional reconstructions, and gross inspection. Scanning electron microscopy was used to study human and rat hearts., Results: In embryonic hearts a prominent horseshoe-shaped myocardial ridge runs from the anterior wall through the apex to the posterior wall of the left ventricle. In the atrioventricular region this ridge is continuous with atrial myocardium and covered with cushion tissue. The anterior and posterior parts of the trabecular ridge enlarge and loosen their connections with the atrial myocardium. Their lateral sides gradually delaminate from the left ventricular wall, and the continuity between the two parts is incorporated in the apical trabecular network. In this way the anterior and posterior parts of the ridge transform into the anterolateral and the posteromedial papillary muscles, respectively. Simultaneously, the cushions remodel into valve leaflets and chordae. Only the chordal part of the cushions remains attached to the developing papillary muscles., Conclusions: Disturbed delamination of the anterior or posterior part of the trabecular ridge from the ventricular wall, combined with underdevelopment of chordae, seems to be the cause of asymmetric mitral valves. Parachute valves, however, develop when the connection between the posterior and anterior part of the ridge condenses to form one single papillary muscle. Thus parachute valves and parachute-like asymmetric mitral valves originate in different ways.

Background: Our previous study of rats exposed for 14 d to microgravity on Cosmos 2044 revealed morphological changes consistent with cardiac atrophy., Methods: In the current comparison study, light and electron microscopic studies were performed on cardiac muscle from 10 rats exposed to hypergravity (continuous centrifugation at 2G) for 14 d., Results: Myofiber area was significantly greater in the 2G papillary muscle as compared with muscle from 10 control rats of the same strain and size. This contrasts with the significant decrease in myofiber area previously seen in the rats exposed to microgravity. At the electron microscopic level, general morphological features were similar in both groups and resembled tissue from control rats from the previous Cosmos studies. However, mitochondria from papillary and ventricular muscle from the 2G rats revealed signs of fatigue typical of the early stage of hypertrophy. These results are consistent with a state of adaptive cardiac hypertrophy for the 2G group.

Background: Altered gravity (G) encountered during spaceflight causes physiologic changes in humans and in experimental animals. In addition to weightlessness (0G) in space, sharply increased G forces are exerted on the spacecraft during the lift-off and reentry phases. Previous studies showed major changes in cAMP-associated activity of rat heart muscle after spaceflight, indicating that (hormone) signaling pathways may have been affected., Hypothesis: The present study was designed to test the hypothesis that cAMP-related cellular responses of exocrine glands after simulated hypergravity (centrifugation at 1.7G) differ from the effects of 0G., Methods: A portion of the parotid and lachrymal gland tissue was fixed for morphologic and immunocytochemical study, and another was used for biochemical determinations. A short-term tissue culture was established from each gland to determine the effects of stimulation by norepinephrine. Heart muscle (ventricle) was also studied. Soluble and particulate fraction extracts of tissue homogenates were prepared, photoaffinity labeled with the [32P]8-N3-analog of cAMP, proteins separated by electrophoresis and the cAMP-reactive proteins (cARP) identified by autoradiography., Results: Differences were seen in protein banding patterns of the gland extracts and in altered cARP distribution in the 1.7G samples of heart ventricle and exocrine gland tissues, when compared with 1G controls. In the heart, cARP increased in the soluble fraction, while the particulate fraction extract showed no change. In acinar cells of the parotid, labeled cARP had accumulated, but decreased after stimulation to the level of the 1G controls. Immunogold labeling showed an increased content of amylase in the secretory granules of the 1.7G animals, while morphologic observation revealed few changes in the structure of parotid acinar cells. The response in the lachrymal gland was translocation of an isoform of cARP from the particulate to the cytoplasmic compartment., Conclusions: Changes distinct from those due to 0G, but specific for hyper-G were found in cARP activity, protein synthesis, as well as in an apparent inhibition of regulated secretion.

The elemental composition of rat cardiac muscle was determined with electron probe x-ray microanalysis (EPMA) of rapidly frozen papillary muscles and trabeculae incubated with ryanodine (1 microM) in either 1.2 or 10 mM [Ca2+]o-containing solutions, paced at 0.6 Hz or tetanized at 10 Hz. Total mitochondrial calcium increased significantly, by 4.2 mmol/kg dry weight during a 7 s tetanus, only in muscles tetanized in the presence of 10 mM [Ca2+]o when cytoplasmic Ca2+ is 1-4 microM (Backx, P. H., W.-D. Gao, M. D. Azan-Backx, and E. Marban. 1995. The relationship between contractile force and intracellular [Ca2+] in intact rat trabeculae. J. Gen. Physiol. 105:1-19). Comparison of total mitochondrial with free mitochondrial Ca2+ reported in the literature indicates that the total/free ratio is approximately 6000 at physiological or near-physiological levels of total mitochondrial calcium. Increases in free mitochondrial [Ca2+] consistent with regulation of mitochondrial enzymes should be associated with increases in total mitochondrial calcium detectable with EPMA. However, such increases in mitochondrial calcium occur only as the result of prolonged, unphysiological elevations of cytosolic [Ca2+].

To determine whether there are differences in cardiac beta-adrenoceptor responsiveness, isoprenaline affinity constants and fractional beta-adrenoceptor occupancy-response relationships for isoprenaline in the early stages of established hypertension, we studied the effects of bromoacetylalprenololmenthane (BAAM) and ([3,5-diamino-6-chloro-N-(1[N-beta-(2-hydroxyl-3-alpha-naphthoxypropy lamino)ethylcarbamoyl]-1-methylethyl)-pyrazine-2-carboxamide (ICI 147 798), slowly reversible beta-adrenoceptor antagonists, on the isoprenaline responses of the left ventricular papillary muscle and the left and right atria of 6-month-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The papillary muscles, but not the right and left atria, of the SHR were less responsive to isoprenaline than those of the WKY. The isoprenaline pD2 values (the negative logarithms of the molar concentrations of agonist producing 50% of the maximum response) were 7.72 and 8.00 on the SHR and WKY papillary muscles, respectively. On the WKY papillary muscle the isoprenaline KA values were 2-3 x 10(-6) M, which is as expected for isoprenaline at beta1 or beta2-adrenoceptors. Isoprenaline had 100-fold greater affinity on the WKY and SHR left atria than on the papillary muscles; the isoprenaline KA values were 2-4 x 10(-8) M. On the WKY papillary muscle and left atrium, isoprenaline had to occupy 3-4% of the beta-adrenoceptors to produce a 50% maximum response; on the WKY papillary muscle and left atrium isoprenaline had to occupy 25-35% and 55%, respectively, of the beta-adrenoceptors to produce a 90% maximum response. The SHR papillary muscles and left atrium had smaller beta-adrenoceptor reserves for isoprenaline than did the WKY tissues. We were unable to obtain isoprenaline KA values on the WKY right atrium. The isoprenaline KA value on the SHR right atrium was 1-4 x 10(-8) M. Because the isoprenaline KA values for the left and right atria are markedly different from those previously reported for isoprenaline at beta1 or beta2-adrenoceptors, we suggest that atypical beta-adrenoceptors might be present on the atria of WKY and SHR. We have also demonstrated a lower beta-adrenoceptor reserve on SHR papillary muscle and atria in the early stages of established hypertension.

The quantitative and qualitative ultrastructural analysis of specialized conducting and working myocytes in papillary muscles of the rabbit heart was made. The volume fractions of myofibers, mitochondria, nuclei, lipids, vacuoles, glycogen, sarcoplasmatic reticulum, T-tubules, lysosomes, and "clear" cytoplasm of conducting and working myocytes were estimated in addition to cell diameters. The results were compared with literature data for the Purkinje cells of different mammals. Principles of morphological description of conducting and working myocytes, allowing to recognize these cells in different parts of the heart and to compare them in different animals, are discussed.

The effects of ruthenium red (RR) on the cellular functions (intracellular Ca2+ handling and contraction) and permeation of the dye through the cell membrane were investigated in intact ferret papillary muscles. The intracellular Ca2+ concentration ([Ca2+]i), measured using aequorin, was simultaneously recorded with tension. The permeation of the dye through the cell membrane was studied with electronmicroscopy. The preparation was continuously stimulated at 0.2 Hz and treated with 50 microM RR at 30 degrees C. [Ca2+]i was increased by electrical stimulation (0.07 and 2 Hz) and rapid cooling (from 30 to 4 degrees C) (RC). In electrical stimulation, RR time-dependently decreased the peak light of aequorin without a significant change in the time course at 30 degrees C. However, in RC, treatment with RR for about 100 min significantly prolonged the decay time of the light signal and increased the peak light. The peak tension in RC was decreased after treatment with RR for a longer time. The pCa-tension relation of skinned preparations was significantly shifted to the right by 50 microM RR. In the RR (50 microM)-treated specimens, mitochondrial outer membranes were darkly stained if OsO4 was used for fixation. Even though the specimen treated with 500 microM RR was fixed without OsO4 and electron staining, the matrices of mitochondria became electron dense. We concluded that RR could penetrate into intact mammalian cardiac myocytes, and that RR inhibits the release of Ca2+ from the sarcoplasmic reticulum in electrical stimulation, inhibits mitochondrial Ca2+ uptake, and decreases the Ca2+ sensitivity of the myofilaments.

The effect of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) and age on inotropic responses of heart papillary muscle to alpha 1-or beta-adrenoceptor stimulation was examined in young (4 months), middle-aged (12 months) and senescent (27 months) male Wistar rats. From the age of two months the rats were fed a diet containing 2% or 12% of fat by weight varying in PUFA type: a) standard low-fat n-6 PUFA diet, b) high-fat n-6 PUFA diet or c) high-fat n-3 PUFA diet. The inotropic responses to alpha 1-adrenoceptor stimulation with phenylephrine were triphasic (positive, negative, then positive). Young, high-fat n-3 PUFA-fed rats exhibited significantly lower negative and higher positive responses to phenylephrine stimulation, and higher positive responses to isoprenaline stimulation than young, high-fat n-6 PUFA-fed rats. On the other hand, no such dietary-related difference was found between young rats fed a high-fat n-3 PUFA diet and a standard low-fat n-6 PUFA diet. The young high-fat n-6 PUFA-fed rats exhibited inotropic responses similar to those of the middle-aged and senescent rats within the three dietary groups. The time to peak force and the time of half relaxation did not differ within dietary and age groups. The findings indicate that dietary n-6 PUFA-rich supplementation at a young age induces changes resembling the effects of age, as evidenced by decreasing cardiac responses to adrenoceptor agonists, such as phenylephrine or isoprenaline.

Nifedipine (20 mg/kg/day) was given to 15-week-old spontaneously hypertensive rats for 20 weeks (SHR-N, n = 8). Comparison was done with sex-matched 15-week-old SHR (SHR-15, n = 7), untreated 35-week-old SHR (SHR-C, n = 10), 15-week-old normotensive Wistar-Kyoto rats (WKY-15, n = 15), and 35-week-old WKY (WKY-15, n = 5). Light and electron microscopic data on the subepicardial, middle and subendocardial layers and papillary muscles of the left ventricle were compared among the five rat groups. In SHR-N, blood pressure was significantly reduced by nifedipine, but was higher than in WKY-35 (199 +/- 11 mmHg vs 121 +/- 13 mmHg). The left ventricular weight/body weight ratio was much lower in SHR-N than in SHR-C, and was even below the baseline value in SHR-15. In addition, cardiac myocyte diameter was much smaller in each myocardial layer of SHR-N than in SHR-C, and was similar to the findings in SHR-15, but still larger than in WKY-35. The interstitial area ratio was markedly reduced in SHR-N and did not differ from that in SHR-15 or even WKY-15, while capillary density was significantly greater than in SHR-C and comparable to that in WKY-35. In SHR-C, large fibrotic foci were common, and many hypertrophic cardiac myocytes showed various degenerative changes including those of mitochondria and widening of the intermyofibrillar spaces. These changes were rarely seen in SHR-N. The intracellular volume ratio of myofibrils did not differ between SHR-N and WKY-35, but was significantly decreased in SHR-C, whereas that of mitochondria did not differ between SHR-N and SHR-C or WKY-35. These findings indicate that despite only a moderate suppression of hypertension, long-term nifedipine treatment caused regression of left ventricular hypertrophy, with cardiocyte hypertrophy, interstitial fibrosis, degenerative changes, and subcellular remodeling being reversed to the baseline levels in SHR-15. In addition, the capillary density was increased to that seen in WKY-35.

A case of severe fetal congestive heart failure due to occlusion of the ductus arteriosus in a mother treated with indomethacin for polyhydramnios and premature contractions is described. Closure of the fetal foramen ovale that escaped detection by prenatal echocardiography was later demonstrated at neonatal autopsy. This case suggests that indomethacin treatment in a ductus-dependent fetus may be hazardous. Therefore, careful surveillance of the fetus exposed to indomethacin in utero is warranted.

Background: The leaflet tissue of the mouse atrioventricular (AV) valves contains a system of wavy collagen bundles that organize like tendons, orientate along lines of tension, and constitute an essential component of the valve tissue. The organization of these bundles is different in the two AV valves, reflecting differences in the anatomy of the entire valvular complex. Further insights into this kind of organization are needed to gain a complete understanding of the functional anatomy of the mouse AV valves., Methods: The endocardial covering of the mouse AV valves (from 21 days to 1 year of age) was eliminated by the sonication or the maceration method. This allowed us to study in situ the organization of the collagenous valve skeleton, as well as the structure of the myotendinous junction., Results: The leaflets of the two AV valves are formed by a fibrous layer (on the ventricular side) and a spongy layer (on the atrial side). The fibrosa is formed by undulating collagen bundles that organize and orientate differently on the right and left sides. The spongiosa is formed, on both sides, by a loose network of thin collagen fibers with no apparent orientation. Myocardial cells in the papillary muscles of the tricuspid valve are elongated and show cone-shaped tips. Collagen fibers attach to the myocyte surface. Collagen struts and thin septa can also be recognized. On the other hand, the collagenous components of the mitral leaflets attach tangentially to the mitral papillary muscles. On the two sides, the myocytes appear to be ensheathed in a layer of collagenous tissue. The sheaths are formed by circularly arranged fibers and appear to be tightly interconnected., Conclusions: The differences in the collagenous organization between the two AV valves reflect differences in the gross anatomy of the valves. The attachment of collagen to the papillary myocytes in the tricuspid valve resembles that of a typical myotendinous junction. However, the collagen-muscle junction in the mitral valve is more similar to the structure of a pennate muscle. The collagen matrix of the heart has been divided into endomysial, perimysial, and epimysial components. The presence of sheaths housing individual myocytes and capillaries, struts, and thin septa, corresponds to the endomysium. The absence of perimysial septa, which aggregate myocytes into groups, is striking, but this may just be a species difference. The appropriateness of the term epimysium, as applied to the heart, is discussed.

Electrically driven rat papillary muscles (1 Hz) were examined for the location of their alpha 1-adrenoceptor and beta-adrenoceptor populations relative to each other. We determined the horizontal position of the dose-response curves for the positive inotropic effects exerted by noradrenaline in the absence and presence of the neuronal uptake blocker cocaine and in the absence and presence of the beta-adrenoceptor antagonist timolol and of the alpha 1-adrenoceptor antagonist prazosin. Cocaine slightly shifted the dose-response curves for alpha 1-adrenoceptor stimulation to a lower concentration of agonist. In contrast, the dose-response curve for beta-adrenoceptor stimulation was markedly shifted by cocaine to a lower concentration of agonist. Experiments with corticosterone (an extraneuronal uptake blocker) revealed no differential shift of either of the dose-response curves. Together, these data indicate that the alpha 1-adrenoceptor population is located more distantly from the adrenergic nerve terminals than the beta 1-adrenoceptor population in rat myocardium.

Quantity and distribution of Purkinje conducting cells, contractile cardiomyocytes, connective tissue, capillaries and nervous structures in the conducting and working myocardium (CM and WM) of the subendocardial area of the anterior papillary muscles of the dog heart were studied. Predominance of the contractile cardiomyocytes volume density over that of Purkinje cells and a higher level of the volume density of capillaries and nervous structures were characteristic of WM comparing to CM while the connective tissue volume density was higher in CM than in WM. These variations in the distribution and quantitative content of tissue structures may determine the primary damage of the subendocardium as well as non-similar response of different animals to the same impact.

beta-Adrenergic stimulation of cardiac muscle leads to an increase in the strength of contraction and an abbreviation of its time course. We have investigated the role of the sarcoplasmic reticulum in these changes by monitoring force and cytoplasmic [Ca2+] in ferret papillary muscles, and the Ca2+ current in isolated ferret myocytes, during the application of isoprenaline in the absence and presence of the sarcoplasmic reticulum inhibitor ryanodine (10(-6) mol/l). Isoprenaline (10(-6) mol/l) led to a marked increase in the size of both the twitch and Ca2+ transient, and a decrease in their duration. In the presence of ryanodine, application of isoprenaline had no significant effect on either the size or the time course of the twitch. However, the increase in the Ca2+ current in response to isoprenaline was the same in the absence and presence of ryanodine. Increasing bathing [Ca2+] led to a prolongation of both the twitch and the Ca2+ transient. In the presence of ryanodine, increasing bathing [Ca2+] still increased the size, but decreased the duration, of the twitch. These data provide direct evidence that both the inotropic and lusitropic effects of isoprenaline are mediated via the sarcoplasmic reticulum.

We developed an optical system for the measurement of the Ca2+ content of sarcoplasmic reticulum (SR) in saponin-treated ventricular muscles of ferrets. After the SR was loaded with Ca2+ by activating the Ca2+ pump of SR, caffeine (50 mM) was applied to release the accumulated Ca2+ from the SR into the bathing solution containing the fluorescent Ca2+ indicator, Fluo-3. As Fluo-3, at high concentrations (approximately 200 microM), predominantly binds most of the Ca2+ released from the SR, the Fluo-3 fluorescence change upon Ca2+ binding gave an estimate of the amount of accumulated Ca2+ in SR before caffeine application. The maximal Ca2+ content of SR, thus estimated, was about 370 mumol/l cytoplasm. The amount of Ca2+ loaded in SR showed bell-shaped dependence on the free Ca2+ concentration ([Ca2+]) of the loading solution, reflecting Ca(2+)-induced Ca2+ release at high [Ca2+] (> or = 1 microM). Mg2+ and H+ decreased the rate of Ca2+ uptake by SR. The present system provides a relatively direct means of measurement of the Ca2+ content of SR, and allows examination of the effects of various interventions on SR Ca2+ uptake, bypassing the large influence of intracellular Ca2+ buffer sites.

In the present study, we analyzed the components of the extracellular matrix (ECM) and its arrangement at the level of the papillary muscles in the adult rat heart using light and transmission and scanning electron microscopy techniques. Our results reveal that after a single dose (6 mg/kg) of dexorubicin to cause a significant decrease and disorganization of the endomysium and perimysium in the first week after injection, affecting the endomysial struts and perimysial strands. Degenerating myocytes and alterations of the coiled perimysial fibers were characteristic 4 weeks after treatment. After 8 weeks, ultrastructural alterations at the level of the plasma membrane of the myocytes and adjacent collagen network were present in the tip of the papillary muscles. These alterations may be responsible for the inefficiency of the valvular apparatus as an initial factor implicated in doxorubicin-induced congestive heart failure. Experimental hypertension, produced by constriction of the abdominal aorta, induced hypertrophy of the left ventricle, with increased perimysium and endomysium of the ECM at the level of the papillary muscles 4 weeks after aortic banding. Interstitial and perivascular fibrosis were observed 8 weeks after surgical treatment, and macrophages around the degenerating myocytes were characteristic 16 weeks after treatment. These alterations of the ECM network have been correlated with their possible implication in ventricular biomechanical properties.

The papillary muscle and chordae tendineae of the rat tricuspid valve were treated with tannic acid-glutaraldehyde fixative and examined with an electron microscope. In the junctional region, the papillary muscle cell tapered abruptly and separated into many processes. One end of bundles of elastic fibers approached these processes or lateral sides of the muscle cell and the other end extended in a longitudinal direction toward the chordae tendineae. Fine networks of elastic fibers were observed in the subendothelial layer of the junctional region.

Intermyocardiocytic fibrosis, i.e., nonreparative interstitial fibrosis with collagen fiber deposition, is commonly found in uremic patients and animals. The volume density of interstitial tissue in the left papillary muscle of uremic animals was found to be increased (from 1.9 +/- 0.7 to 4.2 +/- 1.1%; P < 0.001). The nuclei of interstitial cells, but not of endothelial cells, were enlarged, pointing to an activating signal that specifically acts on interstitial cells. Because of the known action of parathyroid hormone (PTH) on the heart, a potential role of PTH in the genesis of fibrosis was explored by comparing subtotally nephrectomized (NX) parathyroidectomized (PTX) rats receiving by osmotic minipump either saline or rat 1,34 PTH (100 ng/kg per hour dissolved in NaCl). Animals were on a standard 0.95% Ca diet. After PTX, they were switched to a high-calcium (3%) diet. At the end of the 14-day experiments, NX-PTX-PTH animals and NX-PTX-solvent animals were comparable with respect to mean body weight (335 versus 338 g), serum creatinine (1.2 versus 1.2 mg/dL), and serum-Ca (2.66 versus 2.63 mmol/L). The volume densities of cardiac interstitium were 4.71 +/- 0.87 versus 1.49 +/- 0.49, and those of capillaries were 8.07 +/- 1.54 versus 7.94 +/- 2.62, respectively (P < 0.001 by analysis of variance). Thus, PTX abolished and PTH restored intermyocardiocytic changes of experimental uremia. These observations argue for a permissive role of PTH for fibroblast activation and the genesis of the cardiac fibrosis of uremia.