Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
1001 Subcellular Calcium Content in Cardiomyopathic Hamster Hearts In Vivo: An Electron Probe Study Meredith Bond, Abdul-Rahman Jaraki, Candis H. Disch, and Bemadine P. Healy Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 In the Syrian cardiomyopathic hamster heart, abnormal cellular calcium regulation, resulting in cellular calcium overload, is believed to play a role in the pathogenesis of cardiac hypertrophy and failure. Alternatively, the primary abnormality may be coronary vasospasm, resulting in reperfusion-induced necrosis. According to the latter hypothesis, only those cells that suffer an ischemic insult would contain elevated calcium levels. To determine whether a generalized elevation in myocytic calcium exists in myopathic hamster hearts, we measured cellular and subcellular calcium concentrations by electron probe microanalysis in cryosections of 50-day and 96-day myopathic and control hearts, rapidly frozen in vivo. Total calcium content of ventricular homogenates from each group was also measured by atomic absorption spectrophotometry. No significant differences hi subcellular calcium were found by electron probe microanalysis among 50-day and 96-day myopathics and their age-matched controls. In 50-day myopathic and control hearts, mitochondria! calcium was 0.7±0.2 and 0.9±0.2, respectively, and A-band calcium was 3.0±0.4 and 2.6±0.4 nunol calcium/kg dry wt(±SEM). Results from 96-day animals were similar. Localized regions of elevated calcium were found only at sites of necrotic foci: in Na+-loaded cells (mitochondria: 4.7±1.3 (SEM) mmol/kg dry wt), in dying cells (mitochondria: 72±22 (SEM) mmol/kg dry wt) or as extracellular deposits (7-10 mol/kg dry wt). Total calcium content of hearts from myopathic hamsters, as determined by atomic absorption spectrophotometry, was also 13 times (50-day) and 50 times (96-day) higher than controls. These results demonstrate that there is a marked heterogeneity in cellular calcium content in myopathic hamster hearts, but the data do not support the hypothesis of a generalized cellular calcium overload. (Circulation Research 1989;64:1001-1012) N ormal cardiac function depends on the cell's ability to maintain cytosolic calcium (Ca2+) within narrow limits; cardiac dysfunction, on the other hand, is frequently associated with cellular Ca2+ abnormalities.12 The Syrian cardiomyopathic (myo) hamster is a well-studied animal model of idiopathic cardiomyopathy, in which the heart hypertrophies and subsequently develops a dilated type of cardiomyopathy, associated with focal areas of myocardial necrosis, fibrosis, and calcification.*-3 A variety of studies have demonstrated Ca2+ abnormalities to be present from an early, presymptomatic age in myo hamster hearts. Reported changes From the Department of Heart and Hypertension, Research Institute of the Cleveland Clinic Foundation, Cleveland, Ohio. Supported by a Grant-in-Aid from the American Heart Association, Northeast Ohio Affiliate, to M.B. and by the Research Institute of the Cleveland Clinic Foundation. Address for correspondence: Meredith Bond, PhD, Department of Heart and Hypertension, Cleveland Clinic Research Institute, FFb-43, One Clinic Center, 9500 Euclid Avenue, Cleveland, OH 44195-5069. Received July 6, 1988; accepted October 28, 1988. include a decreased activity of the sarcolemmal Ca2+-ATPase67 and decreased Na + ,K + -ATPase activity,7 as well as increased density of voltage sensitive Ca2+ channels,8 although the latter observation is controversial.9 In view of the early onset of many of these changes in Ca2+ metabolism, it has been proposed that a general elevation of intracellular Ca2+ may be an important initiating or aggravating factor in the etiology of the disease. 10 " Alternatively, it has been proposed by Sonnenblick and coworkers12 that the primary defect responsible for development of heart failure in myo hamsters may not be an abnormality in Ca2+ handling by the myocytes themselves but an increased reactivity of the coronary arteries and arterioles. They and others have provided evidence for the existence, in myo hamsters, of coronary vasospasm in situ13 and coronary flow heterogeneity,14 as well as increased reactivity of the microvasculature.13 These investigators predicted that transient ischemia and the reperfusion-induced necrosis that follows would more readily explain the localized, discrete nature 1002 Circulation Research Vol 64, No 5, May 1989 of the observed pathological lesions in myo hearts than a generalized defect in myocytic Ca2+ regulation.12 In contrast to the alternative hypothesis of a generalized Ca2+ overload, reperfusion of populations of ischemic myocytes could result in elevations in cell Ca2+ concentration16-'8 only in localized regions of the heart. Based on this vascular hypothesis, which would result in a focal type of lesion, one would predict the existence of a marked heterogeneity in cellular Ca2+ content in myo hamster hearts, as opposed to a more generalized cellular Ca2+ overload. Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 To date, there have been no direct measurements of cellular or subcellular Ca2+ concentration in myo hamster hearts in situ. Some investigators have measured the total Ca2+ content of ventricular homogenates from myo hearts by atomic absorption spectrophotometry (AA) and found it to be markedly elevated compared with the Ca2+ content of control hearts. 31920 Ca2+ content of isolated mitochondrial fractions has also been shown to be significantly elevated in myo hearts. 1120 The approaches used in these studies are, however, limited in their ability to identify whether the observed elevation in cardiac and/or mitochondrial Ca2+ is a homogeneous phenomenon throughout the heart. An alternative methodology, which avoids the problems associated with measurements of total tissue Ca2+ by AA, is electron probe microanalysis (EPMA) of intracellular and subcellular concentrations of Ca2+ and other elements in cryosections of heart rapidly frozen in situ.2122 In this study, we investigated the question of intracellular Ca2+ overload in myo hamster hearts by directly measuring subcellular Ca2+ content in freezedried cryosections obtained from hearts of myo hamsters and hearts of normal, age-matched controls rapidly frozen in vivo. Using this approach, it has been possible to obtain for the first time direct measurements of subcellular elemental content and elemental distribution in the heart in vivo. Specifically, we sought to determine whether a general elevation in total cytosolic Ca2+ is found in myo hamster hearts 1) during relatively early stages of disease development (50 days) and 2) at a later stage, when significant progression of the disease, including cardiac hypertrophy, has occurred (96 days).4-5 In addition, we investigated whether intracellular Ca2+ overload, if it occurs, is reflected in enhanced mitochondrial Ca2+ uptake. Alternatively, in the absence of a general elevation in basal (diastolic) Ca2+, our objective has been to identify specific localized sites of elevated Ca2+ within myo hamster hearts. Materials and Methods Animals Studied Myo hamsters of the BIO 14.6 strain and agematched control golden hamsters were obtained from Canadian Hybrid Farms, Centerville, Nova Scotia, Canada. Two different age-groups were studied: 44-57 days (mean age, 50 days) and 93-100 days (mean age, 96 days). Rapid Freezing of Hearts In Vivo To rapidly freeze the hamster heart in vivo, a pair of hand-held, mechanically triggered, spring-loaded clamps with freezing surfaces of frozen Freon 22 was used. Their use and operation has previously been described in detail.2223 Briefly, freezing involves filling Al cups, which detach from the ends of the jaws of the clamps, with liquid Freon 22. The Freon is then frozen (freezing point, -156° C) by immersion of the cups, to below the level of the rim, with liquid N2. Just before freezing of the heart in vivo, the cups containing frozen Freon are attached to the ends of the clamps and submerged in liquid N2. Hamsters were anesthetized by intraperitoneal injection of Inactin (80-90 mg/kg body wt) and then taped, dorsal side down, to a board supported over a water bath. Animals were tracheotomized and ventilated with room air at 80-90 breaths/min, with a tidal volume of 1-1.2 ml. To ensure that the ventilation rate and tidal volume used was optimal, in the first few animals, the femoral artery was cannulated, 200-/A1 blood samples were collected in heparinized Clinitubes, and blood pH, Po?, and PCO2 were measured in a Gas Analyzer (Radiometer, Copenhagen, Denmark). The right carotid artery was cannulated with PE-10 polyethylene tubing, and the cannula was then gently inserted past the aortic valve into the left ventricle. Femoral arterial pressure (when measured), left ventricular pressure (LVP), and the first derivative of LVP, dP/ dt, were recorded throughout the experiment, up to the time of freezing of the heart, on a Gould four-channel recorder. (DP/dt was recorded only as an aid to help identify the point in the cardiac cycle at which the heart was frozen.) Once a stable recording of LVP was obtained, the abdomen was opened, the diaphragm cut, and the animal ventilated against positive pressure. A medial incision was made through the sternum, and the ribs were drawn back toward the head to expose the heart. In the earlier hearts that were frozen, the temperature of the water bath was increased to provide a warm and moist environment in the vicinity of the exposed heart. In others, warmed saline (approximately 37° C) was dripped slowly over the surface of the heart. A suture was then placed superficially in the apex of the heart, and a curved piece of Teflon, with a circular hole cut in its center, was gently placed over the heart, so that the heart protruded through the hole; the use of such a "lung pressor" greatly facilitates rapid freezing of the heart without inadvertent freezing of part of the lungs. Hearts were frozen at a specific time point in the cardiac cycle by running the chart recorder at a high speed (50 or 100 mm/sec). The beating heart was then lifted away from the body by the apical suture. The clamps, with attached Al cups containing fro- Bond et al A. : ! •. ! i Uiu J i — si LVP o : ! ' | 1 ! - • • IT - ' •& E o M jJ. s d - . | \ 1003 1 \ \ Calcium in Myopathic Hearts \ - - V T 1^ r '11 I - ! ! ! l|! i I !r. . . Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 zen Freon 22, were held out of the liquid N2 for about 5 seconds to allow a melted layer of Freon to form over the frozen surface.20 The heart was then rapidly frozen by triggering the clamps by hand to rapidly close. The precise time-point of the freeze, within the cardiac cycle, was indicated by an artifact on the LVP pressure trace (Figure 1). Only hearts frozen during diastole were used in this study. The frozen heart, sandwiched between the frozen Freon surfaces of the closed clamps, was quickly cut away from the thorax and rapidly transferred into liquid N2. After removal from the clamps under liquid N2, the atria were chipped away, and the region of the apex at the site of the suture was also removed. Cryoultramicrotomy Two to three pieces of frozen myocardium (approximately 2-3 mm across) were cut from the epicardial surface of the left ventricle of the frozen heart with a liquid Nj-chilled chisel. In some of the hearts from 96-day myo hearts, the piece of myocardium used for cryoultramicrotomy was specifically chosen from regions adjacent to, and including, scarred or fibrotic areas, which have a transmural distribution7 and can be readily identified in the frozen hearts under liquid N2, with the aid of a dissecting microscope. The presence of fibrotic lesions in these areas was later confirmed, at the light microscopic level, using histological sections cut from the same area that had been used for cryoultramicrotomy and EPMA (see below). The small pieces of the frozen heart were then transferred, under liquid N2, to the precooled chamber of a Reichert-Jung FC4D cryoultramicrotome (Cambridge Instrument Co, Deerfield, Illinois) (ambient temperature, -125° C; knife and specimen temperature, -100° C) and glued to Al pins, with the epicardial surface facing up, using toluene (freezing point, -93° C) as a low-temperature cement.24 One FIGURE 1. Examples of left ventricular pressure traces (LVP) and the first derivative ofLVP (dPIdt) from two hamster hearts rapidly frozen with Freon clamps in vivo: in early systole (A) and in early diastole (B). The instant that the heart is frozen is indicated by an artifact (arrow) on the pressure traces. pin with attached frozen myocardium, was then inserted into the microtome chuck and ultrathin cryosections (100-150 nm thick) cut from the surface with a glass knife, with the plane of the sections parallel to the longitudinal axis of the superficial cardiac muscle fibers. Cryosections were transferred dry, within the cooled chamber, using a chilled eyelash probe, onto Ca2+-free carbon support films, prepared as previously described22 on nickel or copper 400-mesh thin bar grids, which were clamped in a Reichert grid holder. The cryosections were then gently pressed flat onto the carbon-coated grids, using a liquid Nrchilled copper rod, and transferred, within the chamber of the cryomicrotome, to a liquid Nr-chilled brass block. The brass block containing the grids was then transferred into an Edwards vacuum evaporator (Rochester, New York) for freeze-drying at or below 10"6 torr overnight. The following day, the freeze-dried cryosections were immediately carboncoated to minimize any possible rehydration from room air and stored under vacuum in a dessicator. Electron Probe Microanalysis Standards and quantitation. Analytical computer programs written in the Somlyos' laboratory at the University of Pennsylvania were adapted for use with an Edax x-ray detector and multichannel analyzer (Edax International Inc, Schaumburg, Illinois). Primary and binary reference standards, together with an absolute sulfur standard (from dried films of 3% bovine serum albumin), were collected. Together with weighting factors obtained from the binary elemental standards, the x-ray spectra collected from albumin allowed the determination of the absolute concentrations of the elements (Na+, Mg2+, P, S, C r , K + , and Ca2+) measured in the cryosections (in miilimoles per kilogram dry weight), as previously described.26 The first and second derivatives of the K+ peaks in the reference spectra were included in the multiple least-squares 1004 Circulation Research Vol 64, No 5, May 1989 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 fit to correct for undetected shifts in detector calibration.27 Absence of systemic errors in the quantitation of intracellular Ca2+ concentrations, was validated by the results of x-ray measurements of the total Ca2+ content of red blood cells (0.13±0.3 [SEM] mmol/ kg dry wt; n=5) in intramyocardial capillaries of control hamster hearts rapidly frozen in vivo. This value is consistent with the very low values previously measured by AA28 and by EPMA.23 Data collection. Samples were transferred into a Philips 400T electron microscope (Pittsburgh, Pennsylvania) using a Gatan temperature-regulated specimen holder (Pleasonton, California). The specimen was then cooled to —100° C for spectral collection. Energy dispersive x-ray spectra were collected from the freeze-dried cryosections in spot mode, at 80 kV, using an Edax 30 mm2 Si(Li) x-ray detector and Edax multichannel analyzer. Spectra were subsequently analyzed on a pdp 11/34 computer (Digital Equipment Corporation, Marlboro, Massachusetts) by a minimum least-squares fit to stored reference spectra.26 Elemental quantitation was carried out using the Hall thin film quantitation procedure.29 X-ray spectra were collected to a standard deviation for Ca2+ of 0.9-1.1 mmol/kg dry wt. Spectra were collected from three cells from each of three to six animals per experimental group and from three separate areas: whole cell (one spectrum per cell), which was an area over several sarcomeres, but not including mitochondria; from mitochondria (two per cell); and from the A band (two per cell). A fifth group consisted of x-ray spectra collected from cells found to be Na+-loaded (K/Na<2) in the vicinity of necrotic foci in cryosections from 96-day myo hearts. Statistical Analysis To distinguish treatment differences from interanimal or cell-to-cell variability as well as variability due to the x-ray counting process itself, x-ray spectra collected from mitochondria, A band, and cell were separately analyzed by nested one-way analysis of variance as previously described.30 If a significant F statistic were obtained, the group or groups showing a significantly different mean Ca2+ content was then identified using a pairwise linear contrast of means. The x-ray data were downloaded from the pdp 11/34 computer onto an AT personal computer and statistical analyses performed using the SAS statistical package (SAS Institute Inc, Cary, North Carolina). Atomic Absorption Spectrophotometry The portion of ventricles not used for cryoultramicrotomy was thawed and wet weight determined. The tissue was dried overnight on tared Al foil in an oven at 105° C and the dry weight then measured. The dried ventricles were then homogenized in a mortar and pestle in a small volume of IN HN0 3 containing 7.2 mM LaCl3. The Ca2+ and Mg2* con- centrations in diluted aliquots of the supernatant (obtained after centrifugation of the HN0 3 acid extract at 700g for 5 minutes) were then measured with a Perkin-Elmer 2380 atomic absorption spectrophotometer (Eden Prairie, Minnesota) using an air acetylene flame. Differences in Ca2+ and Mg*+ content were determined for each age group with an unpaired two-tailed Student's t test. Histology The samples of ventricle that had been mounted on pins for cryoultramicrotomy were saved for histological analysis. The frozen pieces of myocardium on the AJ pins were thawed at room temperature in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, postfixed in OsO4, dehydrated in alcohol and embedded in Epon. Sections 0.5 fim thick were cut on an LKB Ultracut IV microtome (Cambridge Instruments, Ijamsville, Maryland) and stained with a polychromatic stain containing toluidine blue plus basic fuschin.31 Results Rapid Freezing of the Heart In Vivo Figure 1 shows two examples of traces of LVP and dP/dt just before in vivo freezing of hamster hearts in early systole (A) and in diastole (B), respectively. The instantaneous freezing of each heart is clearly indicated by an artifact on the pressure traces. While the heart rate tends to be somewhat slower than in the closed-chested animals, the use of hearts rapidly frozen in vivo, for the determination of subcellular elemental content by EPMA, more closely approximates the natural state of the beating heart in the living animal than does an isolated preparation. The type of ultrastructural preservation achieved by rapidly freezing hamster hearts in vivo with Freon 22 clamps is exemplified in the ultrathin, freeze-dried cryosection shown in Figure 2. Subcellular structures, such as mitochondria, A band, I band, and nucleus are readily identified. The quality of freezing is comparable to that obtained by other investigators in cardiac preparations, such as papillary muscle, rapidly frozen in vitro.32-34 Physiological Parameters Throughout the period of experimental manipulations and surgery, both myo and control hamsters were maintained under optimally oxygenated conditions, with no significant differences in blood gas values between the two strains: blood pH averaged 7.38±0.06(SD), mean PCO2 and PO2 were 38±8 mm Hg and 84±5 mm Hg, respectively (pooling results from both groups). Both 50-day and 96-day myo hamsters weighed approximately 15% less than their age-matched control (Table 1). Both systolic and diastolic blood pressure as well as heart rate, measured in Inactinanesthetized animals, was also significantly lower (p<0.01) in the myo strain at both ages. Bond et al Calcium in Myopathic Hearts 1005 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 — . ^ _ ; FIGURE 2. An example of an ultrathin, freeze-dried cryosection cut from the epicardial surface of a hamster heart rapidly frozen in vivo. M, mitochondria; A, A band; N, nucleus. Total Ca and Mg Content of Ventricles ofMyo and Control Hearts Measured by Atomic Absorption Spectrophotometry The total ventricular Ca2+ content measured by AA in nitric acid digests of 50-day and 96-day myo and control hearts is shown in Figure 3. Ca2+ was found to be markedly elevated in the myo hearts, particularly in the2+96-day-old animals. In contrast, myocardial Mg content did not differ significantly between 96-day myo and control hearts (myo: 38.4±2.1 [SEM] mmol/kg dry wt [n=T\; controls: 39.2±1.3 [SEM] mmol/kg dry wt [«=7]). Among hearts of 50-day myo and control hamsters, Mg2+ content was somewhat higher in the myo group (p<0.02) than in the controls (myo: 41.6±1.2 mmol/ kg dry wt [n=10]; controls: 36.5±1.6 mmol/kg dry wt[n=10]). Cellular and Subcellular Elemental Composition In Vivo Total cellular and subcellular concentrations of Na + , Mg2+, P, S, Cl", K \ and Ca2+ in 50-day and TABLE 1. 96-day myo and control hearts, measured in myocytes of normal ultrastructural appearance away from fibrotic areas are presented in Tables 2-4 (groups 1-4). Group 4 (96-day myo) also includes x-ray spectra from low Na+ cells cut from regions of the ventricle in the vicinity of necrotic foci, as determined by eye under the dissecting microscope, but found not to be immediately adjacent to the pathological lesion by visual inspection of both the cryosections and the plastic embedded sections later obtained from the cutting face of the frozen block. The elemental composition of Na+-loaded cells, in the immediate vicinity of necrotic foci is also shown in Tables 2-4 (group 5). Average values for cellular and subcellular Ca2+ content are summarized graphically in Figure 4. No significant differences in either total cell, A band, or mitochondrial elemental composition were revealed by nested analysis of variance among groups 1-4. In particular, it should be noted that the mean Ca2+ content in all of these four groups, for each of the subcellular Physiological Parameters From 50- or 96-Day Control and Myopathic Hamsters 96±1 day 50±lday Body weight (grams) Systolic BP (mm Hg) Diastolic BP (mm Hg) Heart rate (beats/min) Myopathic (34) Control (26) Myopathic (17) Control (25) 70.9±l.l 126.7+6.9 89.1±5.4 276.0±10.9 84.8±2.3* 168.6+5.9* 112.0+4.5* 349.6+11.7"* IO1.3±2.4 114.8±8.0 8O.4±7.3 273.8±18.1 124.2+2.1* 149.5±5.6* 103.4+4.5* 363.1 + 15.1 BP, blood pressure. Values are mean±SEM. *p<0.01 by two-tailed Student's t test vs. age-matched control. 1006 Circulation Research Vol 64, No 5, May 1989 near fibrotic scars is clearly evident from a comparison of the size of the Ca2+ x-ray peak in an averaged mitochondrial spectrum (A and B) with the Ca2+ peak in an averaged spectrum from mitochondria in myocytes distant from regions of necrosis in 96-day myo hearts (C and D). Nor 50 d •H Myo SO d Nor 9 6 d Myo 96 d 200 150 [C] mmol/kg dry wt • / - SEM 2 100 50 250 300 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 FIGURE 3. Total ventricular Ca * content measured by atomic absorption spectrophotometry (AA) in nitric acid digests of nine 50-day control (nor) and ten 50-day myopathic (myo) hearts and from seven 96-day nor and seven 96-day myo hearts. *p<0.05 vs. 50-day nor; +p<0.01 vs. 96-day nor. regions analyzed, is very low. Thus, we find no evidence of elevated subcellular Ca2+ content in myocytes of normal ultrastructural appearance, away from necrotic foci, in hearts of myo hamsters at either 50 or % days of age. To pursue the cause of the marked elevation in total ventricular Ca content, measured by AA, in myo hearts versus controls (Figure 3), 619 the subcellular composition of myocardium in the immediate vicinity of fibrotic scars (Figure 5) was examined in cryosections from 96-day myo hearts. It was found that cells of normal ultrastructural appearance in the vicinity of these scarred regions were frequently Na+.-loaded (group 5 in Tables 2-4 and Figure 4); the subcellular Ca2+ content of this population of cells (identified by a K+/Na+ ratio of <2:1) was significantly elevated compared with every other group analyzed from myo and control hearts. When compared with group 4, the increase in mitochondrial Ca2+ content in this population of cells was proportionally even greater than the observed elevations in cellular or A band Ca2+, increasing almost fivefold. In Figure 6, the marked elevation in mitochondrial Ca2+ content measured in the Na+-loaded cells TABLE 2. Subcellular Ca Content at Sites of Pathological Lesions Cells within necrotic regions that were clearly irreversibly injured were also identified, as shown in the cryosection in Figure 7, where remnants of Z lines are still evident but crystalline deposits are present in the mitochondria. Mitochondrial Ca2+ in such cells ranged from 21 to 123 mmol/kg dry wt, up to 140 times higher than in normal myocytes, away from fibrotic lesions, in the 96-day myo hearts. An example of an x-ray spectrum from such a Ca2+loaded mitochondrion, showing a marked Ca2+ and P elevation and loss of K + , compared with the spectra shown in Figure 6, is shown in the inset to Figure 7. Other localized sites of highly elevated Ca2+, again found exclusively within necrotic zones, were extracellular Ca2+ phosphate deposits containing 7-10 mol Ca2+/kg dry wt. Discussion Numerous studies have focused on abnormalities of Ca2+ handling as a cause of, or aggravating factor in, the development of a dilated cardiomyopathy in the Syrian cardiomyopathic hamster. Most studies to date, which have shown such abnormalities, have used methods that fail to distinguish focal or regional abnormalities from those that are homogeneous phenomena throughout the heart. For example, a global measure of ventricular Ca2+ content by AA can be significantly affected by the existence of localized regions containing a very high Ca2+ content. Similarly, the mean Ca2+ concentration of a population of isolated mitochondria can be artificially elevated by the contribution of a subpopulation of mitochondria that are Ca2+-loaded.35 In addition, during mitochondrial isolation, Ca2+ uptake can readily occur unless special precautions are taken, such as inclusion of ruthenium red in the isolation medium.36 Total Cell Elemental Content (mmol/kg dry wt ± SEM) Age/Strain 50-day Nor 50-day Myo 96-day Nor 96-day Myo 96-day Myo K/Na<2 No. of Hamsters Total No. of Spectra -4 6 3 4 4 12 14 10 15 8 Na+ ' 169.4±14.7 . 161.2±17.7 132.0±19.9 159.8+16.8 280.5+24.0* Nor, normal (control); Myo, myopathic. *p<0.01 vs. each of the other groups. Mg» P S 57.0±2.4 419.3±23.4 60.7±2.3 65.4±1.8 60.4±3.6 57.3+3.2 461.5±18.8 496.5±21.9 363.9±24.4 388.3±13.4 435.7+13.6 422.1+20.1 391.4±25.0 393.8±14.9 384.9+16.2 cr K+ Ca2+ 119.8+8.7 471.1 ±17.3 561.7±46.2 569.3±38.6 3.4±0.7 2.9±0.5 2.5±0.7 498.5±20.3 3.3±0.5 7.2±1.6* 115.6±14.0 91.7±6.6 110.4±10.1 144.2±11.4 397.4±21.4 Bond et al TABLE 3. Calcium in Myopathic Hearts 1007 Elemental Content of A Band (mmol/kg dry wt ± SEM) Age/Strain 50-day Nor 50-day Myo %-day Nor 96-day Myo 96-day Myo No. of Hamsters Total No. of Spectra 4 6 3 4 4 20 31 22 28 19 S cr K+ Ca2+ 351.8±20.8 368.0+17.3 378.5+22.2 429.2±17.1 428.8+15.0 423.7±15.1 436.1 ±10.6 407.8±13.2 113.5+6.5 131.6±8.7 2.6±0.4 3.0±0.4 97.7±5.3 106.9+6.3 567.5±21.0 688.2±30.5 689.7±43.3 590.9±16.3 3.7±0.8 3.6±0.4 169.5±6.3t 472.3+20.7* 5.3±0.6§ Na+ Mg*+ P 181.5+14.3 181.9±10.7 67.3±2.8 75.6±2.2 159.2±15.4 I68.2±11.9 82.5+3.3 72.2±2.6 68.8±3.2 334.8+16.8* 373.7+17.6 338.5+22.4 Nor, normal (control); Myo, myopathic. *p<0.01 vs. each of the other groups. tp<0.01 vs. %-day Nor. p<0.05 vs. 96-day Myo, 50-day Nor. tp<0.0\ vs. 50-day Myo, 96-day Nor. §p<0.0l vs. 50-day Nor and Myo, p<0.05 vs. %-day Nor and Myo. Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 EPMA, in contrast, allows cellular and subcellular measurements of Ca2+ in rapidly frozen myocardium in situ. Using this approach, the total concentration of Ca2+, and other elements, in subcellular compartments, can be quantified in individual cells with a spatial resolution of up to 10 nm.21 Using EPMA thin film quantitation routines,26-27>» the sensitivity of this technique to small differences in total Ca2+ content, within defined microvolumes of the cell, is of the order of 0.5-0.7 mmol Ca2+/kg dry wt.2*•*> The particular advantages of EPMA over other methods are that 1) subcellular fractionation of the tissue is avoided, and 2) provided that tissue samples are prepared by rapid freezing rather than by conventional aldehyde fixation, the possibility of redistribution of Ca2+ and other diffusible elements is avoided.37 Furthermore, and of particular relevance to this study, 3) regional and cellular variations in the concentration of Ca2+ and other elements can be readily identified by EPMA, since spectra are obtained on a cell-by-cell basis. In this study, a technique has been developed to rapidly freeze hamster hearts in vivo, thus permitting measurement of subcellular elemental content in the intact, blood-perfused heart, without recourse to either tissue isolation or subcellular fractionation and without the use of liquid fixatives, in other words, under conditions that most closely approximate the physiological state of the animal. TABLE 4. Subcellular Elemental Content in Normal Hamster In Vivo The EPMA results obtained from control hamster hearts in the current study show a somewhat higher intracellular Na+ concentration and Ca2+ concentration than EPMA measurements obtained by some other groups from rapidly frozen in vitro preparations. 3233 It is likely that these differences are due to species differences as well as to differences, alluded to previously, between in vitro and in vivo preparations. Specifically, the in vivo heart rate in hamsters (Table 1) is 250-350 beats/min or 4-6 Hz, while in vitro cardiac preparations such as papillary muscle, are generally stimulated at the much lower rates of 0.2-0.3 Hz. Increased rates of beating have been shown to result in intracellular Na + accumulation.38^39 An elevation of cytoplasmic free Ca2+ in diastole, as a direct function of an increased rate of contraction, has also been demonstrated in cardiac myocytes loaded with the Ca2+ sensitive dye, indo I.*0 Since free and bound Ca2+ should be in equilibrium, an elevation in the cytosolic free Ca2+ concentration would result in a higher proportion of cytoplasmic Ca2+ binding sites (e.g., on troponin and calmodulin) being occupied by Ca2+ at any moment. Thus, one might predict that total cytoplasmic Ca2+, as measured by EPMA, would also increase as a function of an increased rate of contraction. Elemental Content of Mitochondria (mmol/kg dry wt ± SEM) Age/Strain 50-day Nor 50-day Myo %-day Nor %-day Myo %-day Myo K/Na<2 No. of Hamsters Total No. of Spectra 4 6 3 4 4 22 37 21 28 16 Na+ %.6+9.8 109.0+9.2 109.9+12.8 104.6±9.2 210.0+14.9* Nor, normal (control); Myo, myopathic. *p<0.01 vs. each of the other groups. tp<0.001 vs. each of the other groups. P 43.6±1.5 42.4+1.3 51.5+2.4 40.8+1.6 44.1+2.7 538.7+17.5 583.5+14.4 571.9+18.4 535.0+9.2 543.5±20.3 S 413.0+13.2 439.8±7.2 443.5±8.4 453.6±8.5 427.0+9.8 cr ++ Ca2+ 76.2+5.6 85.3+5.3 344.6±10.4 440.0+33.0 77.1+5.2 81.1±5.7 125.3+14.0 452.2+28.1 367.6±9.4 348.9±26.9 0.9+0.2 0.7±0.2 1.2+0.4 0.9±0.3 4.7±1.3t 1008 Circulation Research Vol 64, No 5, May 1989 Nor 50 d Myo 50 d Nor 96 d 4 -\, JMITO JABAND 1- 1 lCELL -i H —1 4 Myo 96 d '—u Myo 96 d (K/Na<2) -p 0 2 4 +• 6 1— —1 8 * 4. Average EPMA measurements of the total Ca2* concentration of cell, A band, and mitochondria from 50-day and 96-day myo and control (nor) hamster hearts rapidly frozen in vivo. Only cells with an elevated Na+ concentration "(KlNa <2)" but with normal ultrastructure, in the vicinity of pathological lesions, demonstrated significant elevations in A-band Ca2+, reflected in a marked elevation of mitochondrial Ca2*. *p<0.01 vs. every other group °p<0.0l vs. 50-day myo, nor; +p<0.05 vs. 96-day myo, nor. FIGURE 10 mmol/kg dry wt • / - SEM Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 We believe that it is unlikely that the Ca2+ content measured over the A band includes a contribution from Ca2+ stores in the junctional sarcoplasmic reticulum, as it has been shown, in ultrastructural studies, that the Ca2+ sequestering stores of junctional and corbular sarcoplasmic reticulum are found principally in the regions of the Z line and I band.41 Cardiovascular Function in Cardiomyopathic versus Control Hamsters The lower blood pressure measured in Inactinanesthetized myo hamsters might suggest that cardiovascular function is depressed in prefailure myo hamsters, while the absence of a reflex increase in heart rate, with decreased blood pres- FIGURE 5. Epon embedded sections (0.5 fim) from the epicardial surface of a 96-day myo heart, cut from a region including afibrotic scar. Macrophage infiltration (black arrow) and necrotic myocytes (white arrow; enlarged in the inset) are evident. BV, blood vessel..Magnification, *270; inset, X540. Bond et al n A 4 - si *• 2 c III KKa 3 -- • 0 Jfir f B 400 300 200 : 100 Jl\ lm No } 1 400 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 • 1 2 ] . j 300 200 100 - s Co [Co] •4.7tO3 [Co] •09103 0 JI \ Co 1 0 A sure, may be indicative of an altered baroreflex sensitivity.42 However, no significant differences in blood pressure were noted in conscious myo and age-matched control hamsters at 45 or 150 days (Pierre Wicker, personal communication). It is thus possible that the depressed cardiovascular function observed in 50-day and 96-day anesthetized myo hamsters may be due to different sensitivities of the two strains to anesthesia. Subcellular Elemental Content in Cardiomyopathic Hearts In the majority of myocytes of normal ultrastructural appearance in myo hamster hearts, the intracellular and subcellular elemental content (Na+, Mg2+, P, S, Cl", and Ca2+), measured by EPMA, was not different from that measured in hearts of control hamsters of the same age. Our findings for Na + , Mg2+, P, Cl", and K+ are very similar to the results of Smith and coworkers43 in cryosections from myo and control hearts, rapidly frozen in vitro. However, contrary to their results, we found no evidence for an elevated S content in myo hearts. Since S is much more labile under the electron beam at room temperature than when the specimen is cooled,26 these differences may be due to the fact that our analyses were performed at -100° C while those of Smith and colleagues were carried out at room temperature. In addition, our methods and quantitation routines provide us with the added sensitivity to measure subcellular Ca2+ concentrations, which have not previously been reported in myo hamster hearts prepared by rapid freezing. Contrary to conclusions from previous measurements of either 1) total ventricular Ca2+ content, 2) the Ca2+ content of mitochondrial fractions, and 3) the marked and generalized elevation in the Ca2+ i 1009 KKa D j 1 / 1 Calcium In Myopathic Hearts 1 FIGURE 6. A: Average of 28 x-ray spectra from mitochondria from four 96-day myo hearts rapidly frozen in vivo. B: Averaged x-ray spectrum from A shown at 10 times higher gain and with potassium Ka and Kb peaks stripped by computer. C: Average of 16 spectra from mitochondria in cryosections of 96-day myo hearts from regions of the myocardium immediately adjacent to scarred or necrotic foci. D: Spectrum from panel C with potassium peaks stripped (gain x.10). Mitochondrial Ca1* concentrations are in millimoles per kilogram dry weight±SEM. 1 2 k«V concentration, previously measured by EPMA, in aldehyde-fixed myo hamster hearts,44 our EPMA measurements in cryosections of hearts rapidly frozen in vivo, revealed no evidence of a generalized, inherent intracellular Ca2+ overload at either 50 or 96 days of age. The similarity of the total cytosolic Ca2+ concentration in myo and control hamster hearts is reflected in the equally low mitochondrial Ca2+ content. If, in myo hearts, the free cytosolic Ca2+ concentration were significantly elevated above normal physiological levels, the mitochondrial Ca2+ uptake pathway would be activated,45 resulting in a rapid elevation of mitochondrial Ca2+. Only in myocytes containing an elevated Na + content, in the vicinity of necrotic foci, was an increase in mitochondrial Ca2+ observed. A distinct heterogeneity in cellular Ca2+ content throughout myo hamster hearts, as exemplified by 96-day hearts, was identified in this study. This heterogeneity is compatible with the long-recognized focal nature of myocardial necrosis, fibrosis, and calcification that develop during the course of the disease.46 Our pathological studies confirm the focal nature of the morphological lesions and suggest that Ca2+ overload of myocytes is also focal and closely related to areas of necrosis. This conclusion is consistent with previous EPMA measurements performed on ethanol-fixed tissue from 90-day myo hamster hearts of calcium phosphate-containing mitochondrial granules in myocytes adjacent to interstitial calcified plaques.46 We have also demonstrated in this study that cells, in the vicinity of necrotic or fibrotic lesions, which show no evidence of structural abnormalities, tend to be both Na+- and Ca2+-loaded. Since the cell membrane is normally relatively impermeant to Na + , one possible explanation for these findings may be an increased leakiness of the cell 1010 Circulation Research Vol 64, No 5, May 1989 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 FIGURE 7. Freeze-dried cryosection of cardiac myocyte from necrotic area at epicardial surface of 96-day myopathic heart. Parallel arrows indicate remnants of Z lines in this damaged cell; thick arrow, mitochondria containing an elevated Co3* phosphate content. The x-ray spectrum from this mitochondrion is shown in the inset. membrane. These cells may have suffered a transient ischemic insult or else have an inherent defect in their ability to handle Ca2+, a defect that is present only in some myocytes. Mitochondria, in particular, contained a markedly elevated Ca2+ content in these abnormal cells. Evidence of a heterogeneous Ca2+ distribution has also previously been found in mitochondrial fractions from skeletal muscle of myo hamsters,47 which, like the heart, is characterized by localized regions of cell necrosis. In this latter study, two populations of mitochondria were identified, one fraction with a 50-fold increase in Ca2+ over normal and another larger fraction with Ca2+ concentrations close to normal, indicating that only some areas of the tissue contained Ca2+-loaded cells. It is also of interest to note that, similar to ourfindingsin myocytes near necrotic foci, an increase in intracellular Na+ and Ca2+ and, in particular, an elevated mitochondrial Ca2+ content was measured by EPMA in rabbit papillary muscle that had been reperfused after a period of hypoxia.18 The observation of mitochondrial Ca2+ loading in Na+-loaded cells near necrotic foci in myo hamster hearts indicates that the cytoplasmic free Ca2+ concentration must have increased well above physiological levels, possibly due to an increased permeability of the plasma membrane. The mitochondria! Ca uptake pathway normally exhibits a low affinity for Ca2+ (in cardiac mitochondria the Km is approximately 30 /xM in the presence of 1 mM Mg2*48); however, in the presence of physiological Mg2+ concentrations, the rate of Ca2+ uptake by mitochondria is a sigmoidal function of cytoplasmic Ca2+,49 and thus Ca2+ uptake increases rapidly at supraphysiological Ca2+ levels.45 In 96-day myo hearts, the very high Ca2+ found in dying cells, as well as extracellular Ca2+ phosphate precipitates at sites of pathological lesions, could readily account for the 50-fold higher Ca2+ content of 96-day myo versus control hearts measured by AA. Furthermore, a variability in the number of calcified lesions in myopathic hearts, and thus in the number of regions containing extremely high Ca2+ concentrations, could readily explain the large variability (standard deviation) of AA measurements of total ventricular Ca2+ content in this and previous studies. Since lesions were much less widespread in 50-day than in 96-day myo hearts, as also noted by previous investigators,6-8 we did not attempt to cut cryosections from regions adjacent to necrotic foci in the 50-day hearts. However, confirming previous studies,6-8 we did note in histological sections from 50-day hearts that pathological lesions were present. Thus, we propose that in 50-day, as in 96-day, myo hearts, the large increase in Ca2+ content over age-matched controls, measured by AA, can also account for by the presence of localized calcified lesions. The fact that at 50 days, the Ca2+ content was only 78±24 (SEM) mmol/kg dry wt compared with 251 ±39 (SEM) mmol/kg dry wt in 96-day myo Bond et al Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 hearts confirms the previous findings that lesions are more common in the older animals. In contrast to the elevated Ca2+ content measured by AA in myo hamster hearts, AA measurements of total tissue Mg2+ showed little (50-day) or no (96day) variability between myo and control hearts, as also found by other investigators.19-20 The 14% higher Mg2* content of 50-day myo versus 50-day control hearts is unlikely to be due to differences in the Mg2+ content of the cardiac myocytes themselves, as it was not reflected in significant differences in the EPMA measurements (Tables 2-4). Since AA Mg2+ measurements reflect total Mg2"1" content of a homogenate of the whole ventricle, the differences observed are more likely to arise from differences in Mg2+ content of some other cellular or noncellular component (e.g., red blood cells, vascular smooth muscle cells, or fibroblasts). The fact that AA measurements include a contribution from red blood cells and plasma, both of which contain a relatively low Mg2+ concentration, may also explain the fact that the absolute values of the Mg2+ data obtained by AA were somewhat lower than the Mg2+ concentrations measured by EPMA. In summary, we conclude from this study that there is no evidence for a generalized abnormality in myocytic Ca2+ regulation, leading to cellular Ca2+ overload in prefailure myopathic hamster hearts in diastole. In homogenates of whole ventricle, the elevated Ca2+ content measured in previous studies by AA was confirmed and shown to be attributable to discrete, localized sites of elevated Ca2+ in the regions of necrotic lesions. These findings of a heterogeneous distribution of a Ca2+ abnormality are consistent with the morphological studies that have revealed focal myocardial necrosis, fibrosis, and calcification. Such a focal abnormality could reflect a defect in cellular handling of Ca2+ that affects only some cells of the heart. Alternatively it could support the notion of microvascular spasm leading to focal injury. Acknowledgments The authors wish to thank Dr Andrew Somlyo, University of Pennsylvania, for generous provision of the analytical programs for electron probe microanalysis; Dr James McMahon, Division of Laboratory Medicine, CCF, for use of the analytical electron microscope; Mr Kevin Waters, Department of Musculoskeletal Research, CCF, for computer programming and for computer resource management; Mr Kirk Easely, Department of Biostatistics and Epidemiology, CCF, for help with statistics and Mr Sherwin Parikh for technical assistance. References 1. Schwartz A, Sordahl LA, Entman ML, Allen JC, Reddy YS, Goldstein MA, Luchi RJ, Wybomy LE: Abnormal biochemistry in myocardial failure. Am J Cardiol 1973^32:407-422 2. Ito Y, Chidsey CA: Intracellular calcium and myocardial contractility: IV. Distribution of calcium in the failing heart. J Mol Cell Cardiol I972;4:507-517 Calcium in Myopathic Hearts 1011 3. Bajusz E, Lossnitzer K: A new disease model of chronic congestive heart failure: Studies on its pathogenesis. Trans NYAcadSci 1968,30:939-948 4. Strobeck JE, Factor SM, Bhan A, Sole M, Liew CC, Fein F, Sonnenblick EH: Hereditary and acquired cardiomyopathies in experimental animals: Mechanical, biochemical, and structural features. Ann NY Acad Sci 1979;317:59-93 5. Paterson RA, Layberry RA, Nadkarni BB: Cardiac failure in the hamster: A biochemical and electron microscopic study. Lab Invest 1972,26:755-766 6. Kuo TH, Tsang W, Weiner J: Defective Ca2+ pumping ATPase of heart sarcolemma from cardiomyopathic hamster. Biochim Biophys Ada 1987;900:10-16 7. Panagia V, Singh JN, Anand-Svristava MB, Pierce GN, Jasmin G, Dhalla NS: Sarcolemmal alterations during the development of genetically determined cardiomyopathy. Cardiovasc Res 1984;18:567-572 8. Wagner JA, Reynolds IJ, Weisman HF, Dudeck P, Weisfeldt ML, Snyder SH: Calcium antagonist receptors in cardiomyopathic hamster: Selective increases in heart, muscle and brain. Science 1986;232:515-518 9. Howlett SE, Gordon T: Calcium channels in normal and dystrophic hamster cardiac muscle. Biochem Pharm 1987; 36:2653-2659 10. Lossnitzer K, Janke J, Hein B, Stauch M, Fleckenstein A: Disturbed myocardial calcium metabolism: A possible pathogenetic factor in the hereditary cardiomyopathy of the Syrian hamster. Rec Adv Card Struct Metab 1975 ;6:207-217 11. Wrogeman K, Nylen EG: Mitochondrial calcium overloading in cardiomyopathic hamsters. J Mol Cell Cardiol 1978; 10:185-195 12. Sonnenblick EH, Fein F, Capasso JM, Factor SM: Microvascular spasm as a cause of cardiomyopathies and the calcium-blocking agent verapamil as potential primary therapy. Am J Cardiol 1985^5:179B-184B 13. Factor SM, Minase T, Cho S, Dominitz R, Sonnenblick EH: . Microvascular spasm in the cardiomyopathic Syrian hamster-A preventable cause of focal myocardial necrosis. Circulation 1982;66:342-354 14. Figulla HR, Vetterlein MD, Glaubitz M, Kreuzer H: Inhomogeneous capillary flow and its prevention by verapamil and hydralazine in the cardiomyopathic Syrian hamster. Circulation 1987 ;76:208-216 15. Conway RS, Factor SM, Sonnenblick EH, Baez S: Microvascular reactivity of the myopathic Syrian hamster cremaster muscle. Cardiovasc Res 1987;21:796-803 16. Shen AC, Jennings RB: Myocardial calcium and magnesium in acute ischemic injury. Am J Pathol I972;67:4I7—433 17. Henry PD, Shuchleib R, Davis J, Weiss ES, Sobel BE: Myocardial contracture and accumulation of mitochondrial calcium in ischemic rabbit heart. Am J Physiol 1977; 233:H677-H684 18. Buja LM, Burton KP, Hagler HK: Quantitative x-ray microanalysis of individual myocytes in hypoxic rabbit myocardium. Circulation 1983;68:872-882 19. Crawford AJ, Bhattacharya SK: Excessive intracellular zinc accumulation in cardiac and skeletal muscles of dystrophic hamsters. Exp Neurol 1987;95:265-276 20. Proschek L, Jasmin G: Hereditary polymyopathy and cardiomyopathy in the Syrian hamster II. Development of heart necrotic changes in relation to defective mitochondrial function. Muscle Nerve 1982;5:26-32 21. Somlyo AP: Cell calcium measurement with electron probe and energy loss analysis. Cell Calcium 1985;6:197-212 22. Somlyo AV, Bond M, Silcox JD, Somlyo AP: Direct measurements of intracellular elemental composition utilizing a new approach to freezing in vivo. Proc Electron Microsc Soc Am 1985;18:10-13 23. Somlyo AP, Bond M, Somlyo AV: Calcium content of mitochondria and endoplasmic reticulum in liver frozen rapidly in vivo. Nature 1985^14:622-625 24. Karp RD, Silcox JC, Somlyo AV: Cryoultramicrotomy: Evidence against melting and the use of a low temperature cement for specimen orientation. J Microsc 1982; 125:157-165 1012 Circulation Research Vol 64, No 5, May 1989 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 25. Bond M, Shuman H, Somlyo AP, Somlyo AV: Total cytoplasmic calcium in relaxed and maximally contracted rabbit vein smooth muscle. J Physiol 1984-357:185-201 26. Shuman H, Somlyo AV, Somlyo AP: Quantitative electron probe microanalysis of biological thin sections: Methods and validity. Ultramicroscopy 1976;l:317-339 27. Kitazawa T, Shuman H, Somlyo AP: Quantitative electron probe analysis: Problems and solutions. Ultramicroscopy 1983;! 1:251-262 28. Bookchin RM, Lew VL: Progressive inhibition of the Ca pump and Ca:Ca exchange in sickle red cells. Nature 1980; 284:561-563 29. Hall TA: The microprobe analysis of chemical elements. Phys Tech Biol Res 1971;lA:157-275 30. Bond M, Vadasz G, Somlyo AV, Somlyo AP: Subcellular calcium and magnesium mobilization in rat liver stimulated in vivo with vasopressin and glucagon. J Biol Chem 1987; 262:15630-15636 31. McMahon JT, Ratliff NB: Better methods: 1 fim plastic sections of endomyocardial biopsy specimens within 24 hours. Cor Notes Soc Card Path 1987;2:3 32. Wheeler-Clark ES, Tormey J McD: Electron probe microanalysis of sarcolemma and junctional sarcoplasmic reticulum in rabbit papillary muscles: Low sodium-induced calcium alterations. Circ Res 1987;60:246-250 33. Wendt-Gallitelli M-F, Wolburg H: Rapid freezing, cryosectioning, and X-ray microanalysis on cardiac muscle preparations in defined functional states. J Elect Microsc Tech 1984;1:151-174 34. Johnson D, Isutsu K, Cantino M, Wong J: High spatial resolution spectroscopy in the elemental microanalysis and imaging of biological systems. Ultramicroscopy 1988; 24:221-236 35. LeFurgey A, Ingram P, Mandel LJ: Heterogeneity of calcium compartmentation: Electron probe analysis of renal tubules. J MembrBiol 1986;94:191-196 36. Reinhart PH, van de Pol E, Taylor WM, Bygrave FL: An assessment of the calcium content of rat liver mitochondria. Biochem J 1984;218:415-420 37. Morgan AJ, Davies TW, Erasmus DA: Changes in the concentration and distribution of elements during electron microscope preparative procedures. Micron 1975;6:11-23 38. Lee CO, Dagostino M: Effect of strophanthidin on intracellular Na ion activity and twitch tension of constantly driven cardiac Purkinje fibers. Biophys J 1982;40:185-198 39. Cohen CJ, Fozzard HA, Sheu S-S: Increase in intracellular sodium ion activity during stimulation in mammalian cardiac muscle. Circ Res 1982^0:651-662 40. Lee H-C, Clusin WT: Cytosolic calcium staircase in cultured myocardial cells. Circ Res 1987,61:934-939 41. Sommer JR, Jennings RB: Ultrastructure of cardiac muscle, in Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE (eds): The Heart and Cardiovascular System. New York, Raven Press Publishers, 1986, pp 61-101 42. Higgins CB, Vatner SF, Eckbcrg DL, Brawnwald E: Alterations in the baroreceptor reflex in conscious dogs with heart failure. J Clin Invest 1972;5I:715-724 43. Smith NKR, Morris SS, Richter MR, Cameron IL: Intracellular elemental content of cardiac and skeletal muscle of normal and dystrophic hamsters. Muscle Nerve 1983; 6:481-489 44. Yarom R, Hall TA, Oakley CM: Localized concentrations of elements in hamster cardiomyopathy: Electron microscopic X-ray microanalysis of normal and sick myocardia. Basic Res Cardiol 1977;72:660-670 45. Robertson SP, Potter JD, Rouslin W: The Ca2+ and Mg*+ dependence of Ca2+ uptake and respiratory function of porcine heart mitochondria. 7 Biol Chem 1982;257:1743-1748 46. Burbach JA: Ultrastructure of cardiocyte degeneration and myocardial calcification in the dystrophic hamster. Am J Anat 1987;179:291-307 47. Mezon BJ, Wrogemann K, Blanchaer MC: Differing populations of mitochondria isolated from normal and dystrophic hamster skeletal muscle. Can J Biochem 1974;52:1024-1032 48. Crompton M, Sigel B, Salzmann M, Carafoli E: A kinetic study of the energy-linked influx of Ca2+ into heart mitochondria. EurJ Biochem 1976;69:429-434 49. Scarpa A, Graziotti P: Mechanisms for intracellular calcium regulation in heart: I. Stopped flow measurements of calcium uptake by cardiac mitochondria. J Gen Physiol 1973; 62:756-772 KEYWORDS • cardiomyopathy • electron probe microanalysis • calcium • rapid freezing Subcellular calcium content in cardiomyopathic hamster hearts in vivo: an electron probe study. M Bond, A R Jaraki, C H Disch and B P Healy Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 Circ Res. 1989;64:1001-1012 doi: 10.1161/01.RES.64.5.1001 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1989 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/64/5/1001 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/