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Am J Physiol Heart Circ Physiol 281: H1131–H1136, 2001. Preserved contractile function despite atrophic remodeling in unloaded rat hearts DENISE C. WELSH,1 KONSTANTINA DIPLA,1 PATRICK H. MCNULTY,2 ANBIN MU,1 KAIE M. OJAMAA,2 IRWIN KLEIN,2 STEVEN R. HOUSER,1 AND KENNETH B. MARGULIES1 1 Cardiovascular Research Group, Temple University Medical Center, Philadelphia, Pennsylvania 19140; and 2Division of Endocrinology, North Shore University Hospital, Manhassett, New York 11030 Received 26 January 2001; accepted in final form 18 April 2001 LVAD support, a recent editorial comment by Soloff (31) has raised the valid concern that reduced myocardial mass might limit or offset the potential benefits of LVAD support. Such concerns are amplified by other recent studies demonstrating that cardiac unloading and atrophy produce activation of gene-expression patterns associated with fetal development and pathological hypertrophy (5). In this context, it is reasonable to consider whether myocardial unloading of normal myocardium with associated reductions of muscle mass necessarily result in decreased pump performance. For the purpose of this discussion, we use the term “atrophy” to denote reductions in cardiac and/or myocyte size below normal levels. On the basis of previous studies reporting that atrophy of normal hearts is associated with changes in gene expression resembling those observed with pathological hypertrophy (5, 20, 26), we hypothesized that myocardial unloading would produce concordant decrements in both myocardial mass and function. To test this hypothesis, we unloaded normal myocardium via heterotopic transplantation (HT) in the rat, and performed morphological and functional analyses on isolated myocytes and force-mechanics studies on isolated papillary muscles. cardiac myocytes; heterotopic transplantation; papillary muscles; morphometry METHODS have documented regression of pathological hypertrophy at the cellular level when hearts with advanced dilated cardiomyopathy are supported with a left ventricular assist device (LVAD) for several weeks or months. To date, the changes in myocardial phenotype induced by LVAD support have included regression of abnormal protein expression associated with pathological hypertrophy and failure (1, 2, 33), improvements in distorted myocyte morphology (16, 34), and improvements in myocyte contractility and Ca2⫹ handling (6, 7, 13). Despite these apparently favorable responses to hemodynamic unloading via Animals. Inbred male Lewis rats weighing 275–375 g were used. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). Approval for the studies performed was obtained from the institutional animal care and use committees at Temple University School of Medicine and the North Shore University Hospital. Surgical procedure. Heterotopic heart transplants were performed using a surgical procedure that has been described in detail previously (17, 22). In brief, rats were anesthetized with an injection of pentobarbital sodium (50 mg/kg ip). The chest was opened and the donor heart was exposed. Heparin was administered and the vena cavae and pulmonary veins were ligated. The heart was then excised and flushed with Address for reprint requests and other correspondence: K. B. Margulies, Cardiovascular Research Group, Rm. 805 MRB, Temple Univ. School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140 (E-mail: [email protected]). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. RECENT STUDIES http://www.ajpheart.org 0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society H1131 Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 10, 2017 Welsh, Denise C., Konstantina Dipla, Patrick H. McNulty, Anbin Mu, Kaie M. Ojamaa, Irwin Klein, Steven R. Houser, and Kenneth B. Margulies. Preserved contractile function despite atrophic remodeling in unloaded rat hearts. Am J Physiol Heart Circ Physiol 281: H1131–H1136, 2001.—The present study was designed to determine whether myocardial atrophy is necessarily associated with changes in cardiac contractility. Myocardial unloading of normal hearts was produced via heterotopic transplantation in rats. Contractions of isolated myocytes (1.2 mM Ca2⫹; 37°C) were assessed during field stimulation (0.5, 1.0, and 2.0 Hz), and papillary muscle contractions were assessed during direct stimulation (2.0 mM Ca2⫹; 37°C; 0.5 Hz). Hemodynamic unloading was associated with a 41% decrease in median myocyte volume and proportional decreases in myocyte length and width. Nevertheless, atrophic myocytes had normal fractional shortening, time to peak contraction, and relaxation times. Despite decreases in absolute maximal force generation (Fmax), there were no differences in Fmax / area in papillary muscles isolated from unloaded transplanted hearts. Therefore, atrophic remodeling after unloading is associated with intact contractile function in isolated myocytes and papillary muscles when contractile indexes are normalized to account for reductions in cell length and crosssectional area, respectively. Nevertheless, in the absence of compensatory increases in contractile function, reductions in myocardial mass will lead to impaired overall work capacity. H1132 PRESERVED CONTRACTILE FUNCTION IN ATROPHIC REMODELING AJP-Heart Circ Physiol • VOL Hz using a video edge-detection system (Crescent Electronics; Sandy, UT). Data were stored on a computer for later analysis using Axotape software (Axon Instruments; Foster City, CA). Resting myocyte length, fractional shortening (change in length divided by resting length), time to peak contraction, and time to 50% relaxation were determined in 8–12 cells from each heart. In a subset of 4–6 cells from each heart, the frequency dependence of contraction was assessed by increasing the stimulation frequency from 0.5 to 1.0 to 2.0 Hz. Steady-state fractional shortening was measured at each stimulation frequency. Papillary muscle function. A septal papillary muscle was dissected from 10 normal Lewis rat hearts and 6 heterotopically transplanted donor hearts 2 wk after transplant surgery. Rats were anesthetized and a cardiectomy was performed as described above. The papillary muscles were mounted horizontally at an arbitrary length between metal clips on an isometric strain-gauge transducer in a Plexiglas bath. The chamber was superfused with KHB containing 2.0 mM CaCl2 and equilibrated with 95% O2-5% CO2 at 37°C. Differences in the CaCl2 concentrations used for isolated myocytes and papillary muscles reflect established differences in the dose-response curves for Ca2⫹ between the two preparations (3). The papillary muscle was stimulated with a Grass square-wave stimulator (Grass Instruments; Braintree, MA) at a stimulation voltage 25% above threshold with a 5-ms pulse width. After being mounted, each muscle was equilibrated for 30 min while being stimulated at a frequency of 2 stimulations/min. Muscle length was then gradually and incrementally increased until the muscle attained the length at which maximal developed force was achieved (Lmax). The muscle was then stimulated twice per minute for the next 20 min at Lmax to confirm a stable (⬍10% variation during 20 min) developed force. Twitches from muscles achieving a stable developed force were recorded on a two-channel paperchart recorder (Gould Instruments; Valley View, OH). For experimental measurements, developed force and time to 50% relaxation were calculated for 5–10 consecutive contractions at a frequency of 0.5 Hz. The diameter of each muscle was then measured in place with a calibrated microscope. Cross-sectional area was calculated by assuming the muscle had a cylindrical shape, and developed force was normalized for the cross-sectional area of the muscle. Statistical analysis. All data reported in the text and tables are expressed as means ⫾ SE. Comparisons between heterotopically transplanted and control hearts (in both myocytes and papillary muscles) were analyzed by unpaired Student’s t-test using a commercially available software program (StatView version 4.1; Abacus Concepts; Berkeley, CA). A P value ⬍0.05 was considered statistically significant for all hypothesis testing. RESULTS Morphometric measurements. The results of organ and cellular morphological analyses are presented in Table 1. Compared with normal hearts from agematched rats, 2 wk of hemodynamic unloading via HT was associated with a 23% decrease in the absolute heart weight (P ⬍ 0.001) and a 26% decrease in the heart weight-to-body weight ratio (P ⬍ 0.001). Of note, paired comparisons of the weights of transplanted hearts at the time of transplantation and at explantation 2 wk later demonstrated an average heart mass decrease of 28 ⫾ 1% (P ⬍ 0.001). 281 • SEPTEMBER 2001 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 10, 2017 cold heparinized saline via a catheter in the ascending aorta. The heart was kept in a cold, high-potassium, cardioplegic buffer solution (30) until transplantation. Recipient rats were anesthetized and prepared in the same manner as donor rats. The abdomen was entered through a midline incision and the abdominal aorta and inferior vena cava were exposed and clamped. The donor heart was placed in the right abdominal cavity and end-to-side anastomoses were made between the donor’s aorta and the recipient’s abdominal aorta and between the donor’s pulmonary artery and the recipient’s vena cava. Care was taken to keep the heart cold throughout the surgical procedure, and the total duration of cold ischemia ranged from 45 to 60 min for all allografts. This procedure allows the intact, transplanted heart to be adequately perfused while remaining hemodynamically unloaded. Unoperated Lewis rats served as controls. Myocyte isolation. Myocytes were isolated from seven normal Lewis rat hearts and six heterotopically transplanted hearts 2 wk after heart transplantation. Rats were anesthetized with an injection of pentobarbital sodium (150 mg/kg ip), and a cardiectomy was performed immediately. Each heart was placed on a modified Langendorf perfusion apparatus and perfused with a nonrecirculating, nominally Ca2⫹free solution containing Krebs-Henseleit buffer (KHB) solution [in mM: 12.5 glucose, 5.4 KCl, 1 lactic acid, 1.2 MgSO4, 130 NaCl, 1.2 NaH2PO4, 25 NaHCO3, and 2 sodium pyruvate (pH 7.4)] with 10 mM taurine until the coronaries were clear of blood. The heart was then perfused for 10–15 min with a recirculating digestion solution containing type II collagenase (180 U/ml) and 20 mM taurine. The heart was then removed from the cannula and the right ventricular tissue was removed before the digested left ventricle (LV) was minced in the collagenase solution. The resulting cell suspension was filtered and centrifuged at 25 g. Isolated myocytes were resuspended in a KHB solution containing 1% wt/vol bovine albumin, 10 mM taurine, and 0.5 mM CaCl2. All solutions were equilibrated with 95% O2-5% CO2. The temperature was kept at 37°C throughout the isolation procedure. Myocyte morphometric analysis. Freshly isolated cells were fixed in an isoosmotic solution of 1.5% glutaraldehyde in 0.06 M phosphate buffer. This fixative does not alter cell volume or shape as previously shown by Gerdes and colleagues (12). For each fixed-cell suspension, average myocyte morphological parameters were derived using a combination of cell-volume measurement and isolated myocyte image analysis as previously described (34). Median cell volume was measured in an aliquot containing at least 10,000 cells using a Coulter Channelyzer and a shape factor of 1.05 as previously validated by Hurley (14). Image analysis was performed using the NIH Image Analysis program (version 1.59) and a charge-coupled device camera, frame grabber, and inverted microscope. A total of 60 myocytes were measured from each myocyte suspension, and cells were selected based on visible striations and the absence of membrane blebs or abnormal granularity. For each myocyte preparation, average myocyte cross-sectional area was calculated by dividing median myocyte volume by mean myocyte length. Isolated myocyte function. For analysis of basal myocyte contractility, myocytes were placed in a chamber on the stage of an inverted microscope. The chamber was superfused at 1–2 ml/min with Tyrode solution [containing, in mM: 150 NaCl, 5.4 KCl, 1 CaCl2, 1.2 MgCl2, 10 glucose, 2 pyruvate, and 5 HEPES (pH 7.4)] at 37°C. Myocytes were chosen based on the same criteria as for the morphometric analysis as well as the absence of spontaneous contractions in 1 mM Ca2⫹. Contractions were measured during field stimulation at 0.5 PRESERVED CONTRACTILE FUNCTION IN ATROPHIC REMODELING H1133 Table 1. Heart and myocyte morphology Control Hearts Number of animals Body weight,† g Heart weight, g Heart weight/Body weight,† g/kg Median myocyte volume, m3 Cell length, m Cell width, m Cell cross-sectional area, m 7 315 ⫾ 12 1.12 ⫾ 0.05 3.58 ⫾ 0.12 31,115 ⫾ 1,571 140 ⫾ 12 33 ⫾ 4 225 ⫾ 12 Unloaded Hearts 6 330 ⫾ 15 0.86 ⫾ 0.02* 2.63 ⫾ 0.09* 18,375 ⫾ 1,829 110 ⫾ 5* 26 ⫾ 3* 167 ⫾ 11* Values are means ⫾ SE; * P ⬍ 0.05 vs. control hearts. † Donor rat body weight in unloaded hearts. Fig. 1. Representative steady-state contractions during field stimulation (at 0.5 Hz) of rat ventricular myocytes from heart unloaded with 2 wk of heterotopic transplantation and nonunloaded controls. Cells with equivalent resting cell length (130–133 m) were chosen. Fmax, or time to 50% relaxation in papillary muscles isolated from unloaded transplanted hearts compared with papillary muscles from control hearts. However, absolute Fmax in muscles from transplanted hearts was reduced compared with normal controls. As illustrated in Fig. 3, this reduction was roughly proportionate to the decrease in papillary muscle cross-sectional area that was observed in unloaded hearts. DISCUSSION Cardiac unloading of normal myocardium and pathologically hypertrophied myocardium results in atrophic remodeling at both the organ and cellular levels (1, 4, 8, 9, 17, 19, 27, 34). Data from the present studies indicate that the normal myocardium of heterotopically transplanted rat hearts undergoes atrophic remodeling at the organ and cellular levels as would be expected with the hemodynamic unloading created by this model. This rapid remodeling is clearly shown by the reduced heart weight-to-body weight ratio and the decreased myocyte length, width, and volume in our transplanted rat hearts. However, unlike previous studies with this model, these studies demonstrate Table 2. Myocyte and papillary muscle contractions Control Hearts Isolated myocyte studies Number of cells Fractional shortening, % Time to peak contraction, ms Time to 50% relaxation, ms Isolated papillary muscle studies Number of muscles Cross-sectional area, mm2 Fmax, mg Fmax/area, g/mm2 Time to Fmax, ms Time to 50% relaxation, ms 69 8.9 ⫾ 0.6 163 ⫾ 4 242 ⫾ 7 10 0.186 ⫾ 0.024 268 ⫾ 25 1.70 ⫾ 0.25 152 ⫾ 5 85 ⫾ 2 Unloaded Hearts 60 9.9 ⫾ 0.5 161 ⫾ 6 240 ⫾ 11 6 0.142 ⫾ 0.029* 184 ⫾ 53* 1.55 ⫾ 0.30 164 ⫾ 10 97 ⫾ 8 Values are means ⫾ SE. Fmax, maximal force generation. * P ⬍ 0.05 vs. control hearts. AJP-Heart Circ Physiol • VOL Fig. 2. Stimulation frequency dependence of contraction in fieldstimulated rat ventricular myocytes. Average data from myocytes unloaded with 2 wk of heterotopic transplantation (n ⫽ 30) and from nonunloaded control myocytes (n ⫽ 34) are shown. 281 • SEPTEMBER 2001 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 10, 2017 At the cellular level, 2 wk of hemodynamic unloading produced a 41% decrease in median myocyte volume. This decrease in cell volume reflected both a 21% decrease in average myocyte length (P ⬍ 0.001) and a 26% decrease in average myocyte cross-sectional area (P ⬍ 0.02). These cardiac and cellular morphological findings demonstrate that 2 wk of myocardial unloading via HT is sufficient to produce marked organ and cellular atrophy of the normal heart. Function measurements. The contractile function measurements for isolated myocytes and papillary muscles from normal control hearts and heterotopically transplanted hearts are presented in Table 2. As illustrated in Fig. 1 and summarized in Table 2, compared with normal cardiac myocytes, isolated myocytes from unloaded hearts demonstrated no differences in fractional shortening, time to peak contraction, or time to 50% relaxation at 0.5 Hz. Recognizing that physiological differences might be more apparent during increased stimulation frequency, we examined the fractional shortening of 34 myocytes from control hearts and 30 myocytes from unloaded hearts at stimulation frequencies of 0.5, 1.0, and 2.0 Hz. As shown in Fig. 2, equivalent fractional shortening of control and unloaded myocytes was also observed during progressive increases in stimulation frequency. In isolated papillary muscles, there were no differences in the peak developed force (Fmax)/area, time to H1134 PRESERVED CONTRACTILE FUNCTION IN ATROPHIC REMODELING Fig. 3. Regression plot demonstrating the relationship between the cross-sectional area and maximally developed force (Fmax) in isolated papillary muscle preparations from rat hearts with (E) and without (F) antecedent hemodynamic unloading by heterotopic transplantation. AJP-Heart Circ Physiol • VOL 281 • SEPTEMBER 2001 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 10, 2017 intact contractile function at both the cellular and muscular levels when myocyte shortening is normalized to resting cell length and when papillary muscle force generation is normalized to cross-sectional area. These findings of preserved function using normalized indexes of contractility suggest that atrophic remodeling does not adversely affect sarcomere function under basal conditions. Nevertheless, the striking reduction in absolute Fmax as a result of atrophic remodeling does demonstrate that a net loss of sarcomeres and myocardial mass will reduce myocardial work capacity even when sarcomere function is preserved. Similar to previous studies of in vivo cardiac unloading of the normal heart (4, 8, 9, 17–19), the present studies demonstrated morphological atrophy at the organ and tissue levels. Specifically, we observed a 23% reduction in heart weight and a 27% reduction in heart weight-to-body weight ratio at 2 wk after HT. At the cellular level, we observed that average myocyte volume in unloaded hearts was 41% smaller than in normally loaded control hearts. In addition to demonstrating that organ atrophy is a reflection of cellular atrophy, our cellular morphometrics also demonstrate for the first time that unloading of the normal heart produces balanced reductions in both cellular length and cross-sectional area consistent with reductions in both series and parallel sarcomeres. These proportionate decreases in length and cross-sectional area after unloading of the normal heart are similar to the balanced 20% decreases in myocyte length and width observed by Zafeiridis and colleagues (34) after mechanical unloading with a LVAD in severely failing human hearts (34). However, the present finding contrasts with the disproportionately large (14%) decreases in myocyte cross-sectional area compared with relatively smaller (6%) decreases in myocyte length observed by Gerdes and colleagues (11) after closing a chronic aortocaval fistula in rats. These distinctions may reflect the more complete reductions in both ventricular preload and afterload produced by HT and mechanical circulatory support. The present studies evaluated isolated myocyte contractility in the atrophied heart. These in vitro studies revealed fractional shortening and relaxation re- sponses in myocytes from atrophied hearts that did not differ from those observed in nonatrophied normal controls. By design, functional studies using isolated myocytes assess myocyte contraction and relaxation in the absence of preload or afterload. Our findings demonstrate that load-independent shortening is normal in myocytes with morphological atrophy. As is conventional in such studies, we measured fractional shortening to provide an index of sarcomere shortening. Thus our findings indicate that basic sarcomere function is intact despite morphological atrophy. Because changes in the rates of contraction and relaxation often precede changes in the magnitude of shortening, our findings of normal times to peak contraction and 50% relaxation in unloaded myocytes further attest to preservation of contractile function. Finally, equivalent responses to increasing stimulation frequency also suggest intact contractile function in unloaded myocytes. Beyond the normal cellular contractility that we observed in atrophic myocytes, a recent study by Ritter and colleagues (29) reported supranormal contractility and Ca2⫹ transients in mouse myocytes 5 days after HT. Although distinctions between the present findings and this previous report could reflect species differences or the differences in the duration of unloading, the observations nevertheless support the contention that morphological atrophy need not be associated with impairment of sarcomere function. On the other hand, preliminary observations by Ito and co-workers (15) suggest a slowing of contraction kinetics and decreased contractile reserve in rat myocytes after 5 wk of hemodynamic unloading. Together with the present studies, the studies of Ritter and colleagues (29) and Ito and co-workers (15) suggest possible time-dependent changes in myocyte contractility and contractile reserve in the setting of morphological atrophy with initial increases and subsequent decreases. Despite morphological atrophy evidenced by reduced cross-sectional area, isolated papillary muscles from unloaded hearts exhibited isometric force generation which was equivalent to that observed in normal hearts when Fmax was normalized by cross-sectional area. Because muscle cross-sectional area reflects the number of sarcomeres in parallel, our findings in papillary muscles suggest that basic sarcomere function is intact despite morphological atrophy. In this regard, our findings in isolated papillary muscles are analogous to our findings in isolated myocytes. As in the myocyte studies, the normal time to Fmax and the normal time to 50% relaxation in atrophied papillary muscles further attest to intact contractile performance. Our finding of normal Fmax/area in atrophied papillary muscles under basal conditions is consistent with previous studies by Korecky and co-workers (21) who studied rat isografts 8 wk after HT. A relevant clinical example of the same phenomenon are studies in which LV ejection fraction remained normal while parallel reductions in LV afterload and LV mass were observed in women with anorexia nervosa (32). As in the present study, these previous findings demonstrate that reductions in cardiac workload trigger reductions PRESERVED CONTRACTILE FUNCTION IN ATROPHIC REMODELING AJP-Heart Circ Physiol • VOL also possible that previously reported changes in myosin isoform and glucose metabolism could affect responses to physiological stress without altering basal contractile responses as assessed in the present studies. Ultimately, new studies will be required to determine the conditions under which molecular changes associated with myocardial atrophy are of functional significance. The present studies show that despite morphological atrophy, myocardial unloading via HT is associated with intact contractile function in isolated myocytes and papillary muscles when contractile indexes are normalized to account for differences in cell length and cross-sectional area, respectively. Based on our findings of reduced absolute Fmax, it is likely that previously reported reductions in global cardiac function after HT (8, 9) could simply be a reflection of the reduced cardiac mass. These observations have potential implications for recent observations in diseased human hearts unloaded via mechanical circulatory support. On one hand, improved fractional shortening observed in isolated myocytes (6) should improve overall cardiac contractile performance. On the other hand, the findings in the present study suggest that reductions in organ and cellular mass after mechanical circulatory support (1, 34) may tend to offset gains resulting from improvements in contractile function. Consideration of such potential trade-offs involved in reverse remodeling during mechanical cardiac support may help optimize contractile improvements in failing hearts at both the organ and cellular levels. 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