* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Titin isoform switching is a major cardiac adaptive response in
Survey
Document related concepts
Management of acute coronary syndrome wikipedia , lookup
Coronary artery disease wikipedia , lookup
Heart failure wikipedia , lookup
Cardiac contractility modulation wikipedia , lookup
Electrocardiography wikipedia , lookup
Mitral insufficiency wikipedia , lookup
Jatene procedure wikipedia , lookup
Echocardiography wikipedia , lookup
Myocardial infarction wikipedia , lookup
Hypertrophic cardiomyopathy wikipedia , lookup
Heart arrhythmia wikipedia , lookup
Quantium Medical Cardiac Output wikipedia , lookup
Ventricular fibrillation wikipedia , lookup
Arrhythmogenic right ventricular dysplasia wikipedia , lookup
Transcript
Am J Physiol Heart Circ Physiol 295: H366 –H371, 2008. First published May 23, 2008; doi:10.1152/ajpheart.00234.2008. Titin isoform switching is a major cardiac adaptive response in hibernating grizzly bears O. Lynne Nelson,1 Charles T. Robbins,2 Yiming Wu,3 and Henk Granzier3,4 1 Veterinary Clinical Sciences, College of Veterinary Medicine, 2School of Biological Sciences and Department of Natural Resource Sciences, and 3Veterinary Comparative Physiology and Pharmacology, Washington State University, Pullman, Washington; and 4Molecular Cardiovascular Research Program, Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona Submitted 4 March 2008; accepted in final form 21 May 2008 collagen; bradycardia; diastolic function; echocardiography state of energy conservation in response to harsh climatic conditions. Grizzly bears (Ursus arctos horribilis) do not eat, drink, or urinate and demonstrate minimal activity during their annual 4 – 6 mo hibernation period. Cardiovascular adaptations must occur for the myocardium to remain healthy and efficient during a period of extremely low heart rates and cardiac output (6, 7, 8, 12, 26). Nonhibernators that suffer from bradycardic conditions will develop cardiac chamber dilation due to volume overload during the excessively long diastolic pauses (23, 35, 42). Over MANY MAMMALS UNDERGO A UNIQUE Address for reprint requests and other correspondence: O. Lynne Nelson, Diplomate ACVIM (Internal Medicine and Cardiology), Veterinary Clinical Sciences, Washington State Univ., 100 Grimes Way, Rm. 17, Pullman, WA 99164 (e-mail: [email protected]). H366 time, chamber dilation and elevated end-diastolic pressures lead to congestive heart failure if no intervention is sought (23, 35). However, hibernating grizzly bears tolerate extremely low heart rates without ventricular chamber dilation (30). The mechanisms that circumvent chamber dilation are not well understood, but they might involve increased ventricular stiffness (30). Molecular changes in sarcomeric and extracellular matrix proteins relate to myocardial stiffness in many forms of cardiac disease. In particular, titin, a giant sarcomeric protein (9, 17, 18, 29, 43, 45), and fibrillar collagen types I and III (22, 32, 37, 40) most commonly exhibit changes that affect ventricular diastolic chamber stiffness. Recent studies in animal models of dilated cardiomyopathy and pressure overload have revealed changes in titin isoform expression that increase myocardial stiffness (44, 45). Total titin content is often unchanged, but the increased expression of the stiffer N2B (vs. N2BA) isoform results in higher passive myocardial stiffness (44, 45). Likewise, many chronic cardiac diseases result in an increased accumulation of interstitial collagen I and III via increased gene expression and decreased degradation (22, 33). Collagen metabolism may be stimulated by hemodynamic as well hormonal factors (38, 40). Collagen or titin isoform changes can adversely affect or compensate for increased chamber stiffness, and these proteins could be altered in hibernating bears. We undertook this investigation to evaluate diastolic function and collagen and titin isoform expression in the myocardium of grizzly bears during hibernation compared with bears during the active period. To avoid the confounding effect of anesthesia on cardiac function, we evaluated unanesthetized subjects by echocardiography. We hypothesized that changes in ventricular compliance occur to avoid remodeling the ventricular chamber during hibernation. Such an adaptation could prevent the negative consequences of chronic bradycardiainduced dilation and potential congestive heart failure. MATERIAL AND METHODS Animals. Four three-year-old subadult female grizzly bears were used for this study. The mean weights were 91.7 ⫾ 15 kg in early summer, 120.1 ⫾ 13 kg in late fall, and 95.6 ⫾ 7 kg in late winter, before emergence from hibernation. The animals were housed at the Washington State University Bear Research, Education and Conservation Facility. The animals were maintained according to the Bear Care and Colony Health Standard Operating Procedures, and all 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. 0363-6135/08 $8.00 Copyright © 2008 the American Physiological Society http://www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.32.247 on June 11, 2017 Nelson OL, Robbins CT, Wu Y, Granzier H. Titin isoform switching is a major cardiac adaptive response in hibernating grizzly bears. Am J Physiol Heart Circ Physiol 295: H366 –H371, 2008. First published May 23, 2008; doi:10.1152/ajpheart.00234.2008.—The hibernation phenomenon captures biological as well as clinical interests to understand how organs adapt. Here we studied how hibernating grizzly bears (Ursus arctos horribilis) tolerate extremely low heart rates without developing cardiac chamber dilation. We evaluated cardiac filling function in unanesthetized grizzly bears by echocardiography during the active and hibernating period. Because both collagen and titin are involved in altering diastolic function, we investigated both in the myocardium of active and hibernating grizzly bears. Heart rates were reduced from 84 beats/min in active bears to 19 beats/min in hibernating bears. Diastolic volume, stroke volume, and left ventricular ejection fraction were not different. However, left ventricular muscle mass was significantly lower (300 ⫾ 12 compared with 402 ⫾ 14 g; P ⫽ 0.003) in the hibernating bears, and as a result the diastolic volume-to-left ventricular muscle mass ratio was significantly greater. Early ventricular filling deceleration times (106.4 ⫾ 14 compared with 143.2 ⫾ 20 ms; P ⫽ 0.002) were shorter during hibernation, suggesting increased ventricular stiffness. Restrictive pulmonary venous flow patterns supported this conclusion. Collagen type I and III comparisons did not reveal differences between the two groups of bears. In contrast, the expression of titin was altered by a significant upregulation of the stiffer N2B isoform at the expense of the more compliant N2BA isoform. The mean ratio of N2BA to N2B titin was 0.73 ⫾ 0.07 in the active bears and decreased to 0.42 ⫾ 0.03 (P ⫽ 0.006) in the hibernating bears. The upregulation of stiff N2B cardiac titin is a likely explanation for the increased ventricular stiffness that was revealed by echocardiography, and we propose that it plays a role in preventing chamber dilation in hibernating grizzly bears. Thus our work identified changes in the alternative splicing of cardiac titin as a major adaptive response in hibernating grizzly bears. CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS AJP-Heart Circ Physiol • VOL 13 parameters were assessed for statistical differences. Table 1 lists the echo-derived parameters and the definitions of each. Tissues. Tissues were harvested from 12 animals euthanized for reasons unrelated to this project. All animals were healthy. Six samples were collected for each group (active and hibernating). Each group had three males and three females. The mean age for the hibernating group was nine years. The mean age for the active group was 10.5 years. The age range was 3–21 years in each group. Note that no significant sex difference was found in titin and collagen expression and that no age dependence was detected either (within the available age range). Tissues sampled from active bears were collected during June, July, or August. Tissues samples from hibernating bears were collected in January or February. Sample collecting procedures adhered to guidelines provided by the IACUC, WSU College of Veterinary Medicine. Tissues were collected immediately after euthanization. Samples were obtained from left ventricular free wall (separated into endo-, ecto-, and mesocardium), snap frozen in liquid nitrogen, and stored at ⫺80°C until use. Left ventricular mesocardium was used for this study because it represents the majority of the wall and thus is likely to be the main determinant of left ventricular function. We also analyzed titin isoform expression in the epicardium and endocardium of six bears (4 hibernating and 2 active bears) and found no significant difference between the different layers, unlike the epi-endo gradient that was reported in previous work (5). Thus data from the mesocardium (reported in Titin gel electrophoresis) are likely to well represent the titin expression level in the left ventricular wall. Collagen I and III immunohistochemistry. We used an immunofluorescence method adapted from Neagoe et al. (29). Briefly, left ventricular mesocardium tissues were cut into 6-m sections using a freezing microtome (active, n ⫽ 6; and hibernating, n ⫽ 6). Each sample was processed for both collagen type I and III according to manufacturer’s instructions (US Biological, Swampscott, MA). Specificity for the antibodies was previously validated for numerous species (US Biological). Briefly, the collagen type I antibody (C751017T; US Biological) was diluted 1:200 in PBS. The antibody was added to each section and incubated for 60 min. The samples were washed in PBS for 10 min. FITC-conjugated anti-mouse antibody (11904-79L; US Biological) was added to the sample and incubated for 60 min. The samples were rewashed in PBS for 10 min and then mounted in glycerol containing 1 mM 1,4 phenylenediamine. The same procedure was used for collagen type III antibody (C7510 –39P; US Biological). A negative control was also run for each sample using FITC-conjugated secondary antibody without collagen primary antibody. The samples were examined using fluorescent microscopy (FITC filter, Zeiss Axioskop 2; Peabody, MA) under ⫻63 oil immersion, and images were captured with a digital camera (Zeiss AxioCam; Peabody, MA) as tagged image file format (TIFF) files at 1,016 ms exposure. Images were acquired of each muscle sample of collagen type I, collagen type III, and negative control. Fluorescence was quantified using an NIH-inspired public domain program (ImageJ) (11). The fluorescence quantification was performed by converting the red, blue, and green channels into separate eight-bit grayscale images. The green images were evaluated using the histogram function assessing the color intensity on a scale of 0 to 256. The intensity values were averaged to yield one value for each collagen type and a negative control for each sample. Titin gel electrophoresis. SDS-agarose electrophoresis studies were performed as previously described (44A). Wet gels were scanned and analyzed with one-dimensional scan software (Scanalytics). The integrated optical density of N2BA titin, N2B titin, total titin, and myosin heavy chain (MHC) was determined as a function of the volume of the solubilized protein sample that was loaded (a range of volumes was loaded on each gel). The slope of the linear range of the relation between integrated optical density and loaded volume was obtained for each protein. The N2BA-to-N2B ratio was calculated as the slope of N2BA titin divided by the slope of N2B titin. 295 • JULY 2008 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.32.247 on June 11, 2017 procedures were approved by the Washington State University (WSU) Institutional Animal Care and Use Committee (IACUC) based on the National Institutes of Health (NIH) guidelines. Hibernation began the last week of October when feeding ceased and ended the second week of March when feeding resumed. In early October, food was gradually reduced until completely withdrawn in late October. Water was available ad libitum, and straw was provided for bedding. Bears hibernated in pairs in unheated pens (3 m ⫻ 3 m ⫻ 2.5 m) with continuous access through a small door to an outdoor area (3 m ⫻ 5 m ⫻ 5 m) that was covered on all sides to minimize external noise and stimulation. Because the pens were open to the outside, the bears experienced daily light and temperature fluctuations. The four bears were hand raised, human socialized, and trained for echocardiography. The bears have a comfortable routine of handling and were fully conscious for this study. These unanesthetized bears were used to avoid the potential confounding effects of anesthesia on in vivo assessment of cardiac function parameters by echocardiography. Diastolic function was measured in all the bears of the study at three different periods during the active phase of the year (June, July, and August) and the hibernation period (December, January, and February). Echocardiography. All bears underwent a complete transthoracic echocardiographic examination that included two-dimensional, Mmode, spectral, and color-flow Doppler evaluations. Echocardiography was performed by the same person (O. Lynne Nelson) using commercially available equipment (Siemens Acuson Ultrasound; Cypress, Bothell, WA). The bears were imaged in sternal recumbency with forelegs positioned cranially to optimize the parasternal thoracic windows. Standard imaging planes (canine) and function calculations have been previously described (37) and were performed in accordance with the American Society of Echocardiography (4, 15, 34). Video images were captured using a commercially available digital echocardiography software program, and data was collected offline using a workstation (Siemens Acuson Ultrasound; Cypress). The following echocardiographic parameters were collected: end-diastolic volume index (milliliters), the maximum diastolic volume by modified Simpson’s rule divided by the body mass in kilograms; stroke volume index (milliliters), volumetric calculated stroke volume divided by body mass in kilograms; percent left ventricular ejection fraction, volumetric calculation of left ventricular ejection by modified Simpson’s rule; cardiac index (milliseconds per minute per kilogram), cardiac output divided by body mass in kilograms; left ventricular muscle mass (grams), calculated by area length method; left ventricular end-diastolic volume-to-left ventricular mass ratio, end-diastolic volume divided by left ventricular muscle mass; left ventricular wall relaxation time (milliseconds), the time recorded for mechanical relaxation as identified by the left ventricular free wall excursion on the M-mode exam (point of maximal excursion in systole to first point of minimal excursion in diastole); left ventricular wall relaxation rate (meters per second), the rate of mechanical relaxation as identified by the slope created by connecting the two points above; left ventricular isovolumic relation time (milliseconds), the time from aortic valve closure to mitral valve opening; left ventricular early filling-to-atrial contraction ratio, the ratio of the velocity of left ventricular early filling to the velocity of atrial contraction; deceleration time of early left ventricular filling (milliseconds), time required for maximal left ventricular filling velocity to decelerate to baseline; pulmonary venous systolic-to-diastolic flow velocity ratio, maximal pulmonary venous systolic velocity divided by maximal diastolic velocity; and pulmonary venous diastolic flow deceleration rate (meters per second), time required for maximal pulmonary venous diastolic flow velocity to decelerate to baseline. The mean of four consecutive measurements was considered average for each parameter during each data collection period. To account for differences in body weight, volume parameters were normalized to body weight (American Society of Echocardiography Index of Published Guidelines, http:// asecho.org/Guidelines.php). Of the echocardiography data collected, H367 H368 CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS Statistical analysis. Analysis was performed using commercial statistical software (version 15, Minitab statistical software). Repeatedmeasures ANOVA was used to compare echocardiography parameters and collagen quantification among the two groups, active and hibernating bears. Assumptions of ANOVA were satisfied; a P value of ⬍0.05 was considered significant. Because of the large number of echocardiography parameters, and possible multiplicity issues therein, a Bonferroni adjustment was made. The stricter P value threshold of ⱕ0.003 was considered significant for the echocardiography parameters. Paired t-tests were conducted to evaluate differences in total titin and N2BA-to-N2B ratios between hibernating and active bears. A P value of ⬍0.05 was considered significant. All data are presented as means ⫾ SD. RESULTS DISCUSSION Our goal was to study cardiovascular adaptations that allow the myocardium to remain healthy and efficient during a period of extremely low heart rates and cardiac output. Our study is the first that uses unanesthetized active and hibernating grizzly bears to avoid the confounding effects of drugs on cardiac function. For example, a previous study on anesthetized bears revealed opposing results in ejection fraction (decreased in hibernation), and heart rate, compared with those of the unanesthetized bears. Systolic and diastolic function parameters Table 1. Echocardiographic parameters recorded in conscious grizzly bears during summer active and hibernation period Parameters Active Hibernation P Value Heart rate, beats/min End diastole Interventricular septal wall thickness, cm Left ventricular free wall thickness, cm Left ventricular internal dimension, cm End-diastolic volume, ml End-diastolic volume index, ml/kg Stroke volume, ml Stroke volume index, ml/kg Left ventricular ejection fraction, % Cardiac index, ml 䡠 min⫺1 䡠 kg⫺1 Left ventricular wall relaxation rate, m/s Left ventricular wall relaxation time, ms Left ventricular isovolumic relaxation time, ms Left ventricular inflow early filling-to-atrial contraction ratio Deceleration time of early ventricular filling, ms Pulmonary venous systolic-to-diastolic flow velocity ratio Pulmonary venous diastolic flow deceleration rate, m/s 83.8⫾12 18.7⫾8.4 ⬍0.0001 1.5⫾0.04 1.3⫾0.08 6.0⫾0.09 219.3⫾15.6 2.4⫾0.14 127.5⫾4.5 1.4⫾0.04 66.4⫾5.2 117.7⫾14.2 0.09⫾0.02 123.3⫾24 63.4⫾10.7 1.4⫾0.2 143.2⫾20.3 1.1⫾0.3 3⫾0.7 1.3⫾0.07 1.1⫾0.1 5.6⫾0.3 188.9⫾6.2 1.9⫾0.13 110.3⫾6.4 1.2⫾0.02 65.8⫾3.9 33.6⫾6.1 0.04⫾0.01 268⫾38.8 97.4⫾15.7 4.9⫾1.1 106.5⫾14.6 0.4⫾0.06 1.4⫾0.3 0.008 0.05 0.196 0.06 0.710 0.03 0.968 0.736 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.003 0.002 ⬍0.0001 0.003 Values are means ⫾ SD; n ⫽ 4 grizzly bears during the summer active and 4 grizzly bears during the hibernation periods. Note that to account for differences in the size of the bears, various indexes are shown normalized to body mass. A P value of ⱕ0.003 was considered significant. End-diastolic volume index is the diastolic volume ⫼ body mass in kilograms; stroke volume index is the stroke volume ⫼ body mass in kilograms; and cardiac index is the cardiac output ⫼ body mass in kilograms. AJP-Heart Circ Physiol • VOL 295 • JULY 2008 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.32.247 on June 11, 2017 Echocardiography. Hibernating bears had significantly lower heart rates (19 vs. 84 beats/min) and cardiac output indexes (33.6 vs. 117.7 ml䡠min⫺1 䡠kg⫺1) than the active bears (Table 1). (Note that the heart rates given for mean rates were an average of 6 measures during the course of the echocardiogram. The heart rate that was used to calculate cardiac output was the instantaneous rate at the time of the Simpson’s calculation. Because of the fact that the bears have sinus arrhythmia during hibernation, the mean heart rate and the rate used for cardiac output were not exactly identical.) We found that the end-diastolic volume index, stroke volume index, and left ventricular ejection fraction were not different between hibernating and active bears (Table 1). The diastolic volume-to-left ventricular muscle mass ratio was significantly greater in hibernating bears (Fig. 1A). Thus total volume was not different between the two groups, but left ventricular muscle mass was significantly lower (Fig. 1B). The left ventricular wall relaxation rate was decreased and the left ventricular and isovolumic relaxation times were longer, but the deceleration time of early ventricular filling was shorter in hibernating bears (Fig. 2). The ratio of early ventricular filling to late ventricular filling of the atrial contraction was significantly increased in hibernating bears. The ratio of pulmonary venous systolic to diastolic flow was significantly lower and the pulmonary venous diastolic rate was significantly faster in hibernating bears (Fig. 3). Thus heart rate and relaxation rates were slower, but early ventricular filling ended sooner in hibernating bears, indicating increased ventricular stiffness. Collagen I and III. The mean collagen type I values were 36.7 ⫾ 6.0 in hibernating bears and 33.9 ⫾ 4.3 in active bears (P ⫽ 0.4). The mean collagen type III values were 48.7 ⫾ 11.6 in hibernating bears and 45.7 ⫾ 7.2 in active bears (P ⫽ 0.6). Collagen type I and III comparisons were not statistically significant between the two groups of bears. Likewise, the ratio of collagen type I to collagen type III was not significantly different between groups. The mean ratio in hibernating bears was 0.8 ⫾ 0.2 compared with 0.7 ⫾ 0.2 (P ⫽ 0.79) in active bears. Titin protein expression. High-resolution SDS-agarose gels revealed prominent N2B and N2BA bands in both active and hibernating bears. The densitometric gel analysis revealed no difference in the total titin-to-MHC ratio between the two groups (active, 0.19 ⫾ 0.06; and hibernating, 0.18 ⫾ 0.02; P ⫽ 0.7). The mean ratio of N2BA to N2B titin was 0.73 ⫾ 0.07 in the active bears and decreased significantly to 0.42 ⫾ 0.03 (P ⫽ 0.006) in the hibernating bears (Fig. 4). Thus total titin was not different between active and hibernating bears, but the mean expression of the stiffer N2B isoform was increased at the expense of the more compliant N2BA isoform. CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS H369 Fig. 1. Left ventricular (LV) end-diastolic volume-to-muscle mass ratio (milliliters/ grams; A) and muscle mass (grams; B) in active and hibernating conscious bears (n ⫽ 4). Total volume was not different between the 2 periods, but LV muscle mass was significantly lower. All data are presented as means ⫾ SD. P ⱕ 0.003, significant. Fig. 2. LV early filling deceleration time in an active (A) and hibernating (B) bear. a, ventricular filling waveform due to atrial contraction; DTE, deceleration time of early ventricular filling waveform; E, early ventricular filling waveform. Note: in the hibernating bear with profound bradycardia, the atrial contraction waveform seen is from the previous diastolic cycle. AJP-Heart Circ Physiol • VOL disease (27). Higher ratios are typically associated with elevated myocardial wall stress and are known to trigger compensatory ventricular muscle hypertrophy (31). Instead, reduced cardiac mass is present in hibernating bears, which might be due to the absence of food intake during hibernation or decreased activity. Diastolic function parameters revealed prolonged relaxation times during hibernation and reduced early left ventricular filling waveform deceleration time (DTE). Increased relaxation times have been associated with bradycardia (32), but the reduced DTE that we found is unique (Fig. 2). DTE is known to be an accurate indicator of ventricular stiffness in numerous cardiac conditions and is a clinically accepted standard measure of left ventricular operating stiffness (4, 15). The restrictive filling pattern of pulmonary venous inflow in hibernating bears was also consistent with increased ventricular stiffness (Fig. 3), which mirrored the early ventricular filling waveform and DTE. Chamber stiffness relates to how much pressure is required to fill the ventricle for a given volume of fluid. The stiffer the chamber, the more rapid the pressure rises inside the chamber and the earlier the filling phase is completed (2, 3, 15). Left ventricular stiffness is governed by a number of factors that include ventricular geometry (particularly hypertrophy) and myocardial stiffness (largely related to tissue Fig. 3. The ratio of pulmonary venous systolic to diastolic flow (PVs/d) was significantly lower in hibernating bears (B). A: active bear. The reduced PVs/d is consistent with a hemodynamic restrictive ventricular filling pattern. S, atrial filling from the pulmonary venous system during cardiac systole; D, atrial and ventricular filling from the pulmonary venous system during cardiac diastole (early filling). 295 • JULY 2008 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.32.247 on June 11, 2017 are known to be affected by anesthetics (19, 36). Even though the use of unanesthetized bears is challenging, it is clear that the echocardiography of conscious, well-adjusted subjects more accurately represents true cardiac physiology. As expected, the hibernating bears had significantly lower heart rates and cardiac output than active bears (Table 1). Ventricular volumes were not different from the active period to hibernation, but left ventricular muscle mass was significantly lower during hibernation (Fig. 1). Because ventricular mass returns to baseline during the next active period (unpublished data), it is possible that cardiac myocytes atrophy during hibernation and hypertrophy during the active period. However, myocyte death and regeneration might participate to some extent since these cellular processes have been documented in certain pathological states (1). The finding of reduced cardiac mass (⬃26% change) during hibernation is in contrast to that of skeletal muscle, which demonstrates limited atrophy (⬍10% change) with disuse (24, 39). These findings suggest interesting differences between adaptations in cardiac versus skeletal muscle that warrant future follow-up studies highlighting an independent measure of cardiomyocyte size. Systemic arterial pressures were not obtained in these conscious bears, but blood pressure did not differ between active and hibernating anesthetized bears in an earlier study (30). The ventricular volume-to-mass ratio is an important clinical determinant of ventricular wall stress in humans with cardiac H370 CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS collagen content and titin expression) (4, 9, 17, 18, 33). With the consideration that myocardial hypertrophy is not detected in hibernating bears, it is likely that the increased ventricular stiffness involves changes in either collagen or titin. Our findings indicate that collagen expression was not different between active and hibernating bears, suggesting that collagen played no major role in altering ventricular stiffness characteristics. We found no difference in the total titin-to-MHC ratio between the two groups, but the mean expression of the stiffer N2B isoform was increased at the expense of the more compliant N2BA isoform in hibernating bears (Fig. 4), indicating that changes in titin expression might explain the increased ventricular stiffness. Preliminary data suggest that ⬃1 wk after feeding starts, at the beginning of the active period, echo parameters have mostly recovered to those of active bears. However, additional data are needed to confirm and extend this result, including exploring the role titin and collagen in the recovery process. Hibernating rodents have demonstrated opposing results in titin expression (6, 43). Some of the differences between our study and that of hibernating rodents may be due to conditions of study (microgravitation for rodents), difference between cardiac and skeletal muscle adaptations, or possibly due to species differences. For example, in contrast to hibernating ground squirrels, hibernating bears do not show degradation of fast muscle fibers (20). Hibernating bears are also unique in that they are less hypothermic (33°C) than hibernating rodents (5°–7°C) (21). Thus calciumhandling dynamics may be affected to a lesser degree in bears than in rodents. Titin is a giant elastic protein that spans form Z-disk to M-line. The I-band region of titin is extensible and functions as a molecular spring that develops passive force in sarcomeres that are stretched beyond their slack length. The difference in the molecular mass of N2BA and N2B isoforms (⬃3.3 vs. ⬃3.0 MDa) is largely due to the differential splicing of the tandem Ig and the PEVK segments of titin that comprise the AJP-Heart Circ Physiol • VOL ACKNOWLEDGMENTS We thank Caroline Benoist, Abbey Burgess, Jennifer Fortin, Gary Radamaker, Justin Teisburg, and Pam Thompson for excellent technical assistance. Present address of Y. Wu: Dept. of Internal Medicine, Univ. of Iowa, Iowa City, IA. GRANTS This work was supported by the Autzen Foundation, the Washington State University Bear Research Center, and National Heart, Lung, and Blood Institute Grant HL-062881. 295 • JULY 2008 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.32.247 on June 11, 2017 Fig. 4. SDS-agarose gel electrophoresis of titin expression in active (n ⫽ 6) and hibernating (n ⫽ 6) grizzly-bear hearts. N2BA-to-N2B expression ratio is significantly lower in hibernating bears. A, active period; H, hibernating; MHC, myosin heavy chain. molecular spring. As a result, the molecular spring is shorter in N2B titin, and a given change in sarcomere length gives rise to a titin-based force that is higher for N2B titin (its shorter extensible region results in higher fractional extensional) than for the longer N2BA isoform (9). Thus the upregulation of N2B titin can explain the increased wall stiffness in hibernating grizzly bears. Correlations between titin expression and ventricular systolic and diastolic function have been observed in humans (14, 28). In dilated cardiomyopathy, increased N2BA expression resulted in a decrease in ventricular stiffness, which may be important in reducing ventricular filling pressures for a given volume (reducing symptoms of congestive heart failure) (3). However, although diastolic function improved, increased N2BA expression has been correlated with a negative effect on systolic function (14, 16). This can be explained by the finding that titin-based passive force enhances calcium sensitivity. Thus the calcium sensitivity effect is reduced when N2BA-toN2B ratios are high (14, 25). In contrast to human dilated cardiomyopathy, an increased calcium sensitivity is predicted in hibernating grizzly bears due to the upregulation of N2B titin. Changes in titin isoform expression might be an adaptive response that occurs during hibernation to allow the myocardium to remain healthy and efficient during a period of extremely low heart rates and cardiac output. A possible explanation for the functional significance of increased ventricular stiffness may be that it is compensatory for the reduced muscle mass relative to volume and the profound bradycardia. Increased ventricular wall stress and prolonged diastolic filling are typically associated with dilation/remodeling of the chamber. A chamber that is stiffer is less likely to distend or dilate. Increases in ventricular stiffness would have to be well balanced with rising ventricular and atrial pressures in diastole. If diastolic filling pressures increased greatly, congestion would occur. There is no evidence of increased left atrial pressures in hibernating bears since the left ventricular isovolumic relaxation period is prolonged (this period is classically shortened with high atrial pressures). Diastolic dysfunction and congestive failure become particularly relevant with exercise and activity (10). Thus inactivity in hibernating bears might promote tolerance of increased ventricular stiffness. If the lower N2BA-to-N2B ratios are associated with enhanced calcium sensitivity and systolic function as in humans, it then follows that any decline in diastolic function might be offset by improved systolic function during hibernation. Thus we propose that changes in alternative splicing of cardiac titin constitute an adaptive response in hibernating grizzly bears. CARDIAC TITIN ISOFORM SWITCHING IN HIBERNATING BEARS REFERENCES AJP-Heart Circ Physiol • VOL 25. Makarenko I, Optiz CA, Leake MC, Neagoe C, Kulke M, Gwanthmey JK, del Monte F, Hajjar RJ, Linke WA. Passive stiffness changes caused by upregulation of compliant titin isoforms in human dilated cardiomyopathy hearts. Circ Res 95: 708 –716, 2004. 26. Milsom WK, Burlington RF, Burleson ML. Vagal influence of heart rate in hibernating ground squirrels. J Exp Biol 185: 25–32, 1993. 27. Mirsky I. Assessment of diastolic function: suggested methods and future considerations. Circulation 69: 836 – 841, 1984. 28. Nagueh SF, Shah G, Wu Y, Torre-Amione G, King NM, Lahmers SL, Witt CC, Becker K, Labiet S, Granzier HL. Altered titin expression, myocardial stiffness, and left ventricular function in patients with dilated cardiomyopathy. Circulation 110: 155–162, 2004. 29. Neagoe C, Kulke M, del Monte F, Gwathmey JK, de Tombe PP, Hajjar RJ, Linke WA. Titin isoform switch in ischemic human heart disease. Circulation 106: 1333–1341, 2002. 30. Nelson OL, McEwen MM, Robbins CT, Felicetti L, Christensen WF. Evaluation of cardiac function in active and hibernating grizzly bears. J Am Vet Med Assoc 223: 1170 –1175, 2003. 31. Olivetti G, Capasso JM, Meggs LG, Sonnenblick EH, Anversa P. Cellular basis of chronic ventricular remodeling after myocardial infarction in rats. Circ Res 68: 856 – 869, 1991. 32. Pavlik G, Olexo Z, Sido Z, Frenkl R. Doppler echocardiographic examinations in the assessment of the athletic heart. Acta Physiol Hung 86: 7–22, 1999. 33. Salazar BL, Albeniz SR, Guedon TA, Miqueo AG, Querejeta R, Martinez JD. Altered fibrillar collagen metabolism in hypertensive heart failure. Current understanding and future prospects. Rev Esp Cardiol 59: 1047–1057, 2006. 34. Schiller NB, Shah PM, Crawford M, DeMaria A, Devereux R, Fiegenbaum H, Gutgesell H, Reichek N, Sahn D, Schnittger I, Silverman NH, Tajik AJ. Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on quantitation of twodimensional echocardiograms. J Am Soc Echocardiogr 2: 358 –367, 1989. 35. Schoenmakers M, Ramakers C, van Opstal JM, Leunissen JDM, Londono C, Vos MA. Asynchronous development of electrical remodeling and cardiac hypertrophy in the complete AV block dog. Cardiovasc Res 59: 351–359, 2003. 36. Stein AB, Tiwari S, Thomas P, Hunt G, Levent C, Stoddard MF, Tang XL, Bolli R, Dawn B. Effects of anesthesia on echocardiographic assessment of left ventricular structure and function in rats. Basic Res Cardiol 102: 28 – 41, 2007. 37. Thomas WP, Gaber CE, Jacobs GJ, Kaplan PM, Lombard CW, Moise NS, Moses BL. Recommendations for standards in transthoracic two-dimensional echocardiography in the dog and cat. J Vet Intern Med 7: 247–252, 1993. 38. Timonen P, Magga J, Risteli J, Punnonen K, Vanninen E, Turpeinen A, Tuomainen P, Kuusisto J, Vuolteenaho O, Peuhkurinen K. Cytokines, interstitial collagen and ventricular remodeling in dilated cardiomyopathy. Int J Cardiol 10: 1– 8, 2007. 39. Tinker DB, Harlow HJ, Beck TD. Protein use and muscle-fiber changes in free-ranging, hibernating black bears. Physiol Zool 71: 414 – 424, 1998. 40. Tokuda K, Kai H, Kuwahara F, Yasukawa H, Tahara N, Kudo H, Takemiya K, Koga M, Yamamoto T, Imaizumi T. Pressure-independent effects of angiotension II on hypertensive myocardial fibrosis. Hypertension 43: 499 –503, 2004. 42. Verduyn SC, Ramskers C, Snoep G, Leunissen JD, Wellens HJ, Vos MA. Time course of structural adaptation in chronic AV block dogs: evidence for differential ventricular remodeling. Am J Physiol Heart Circ Physiol 280: H2882–H2890, 2001. 43. Vikhlianstsev IM, Malyshev SL, Shenkman BS, Podlubnania ZA. The behavior of titin and the proteins of its family from skeletal muscles of ground squirrels (Citellus undulatus) during hibernation and rats under conditions of simulated microgravity. Biofizika 49: 995–1002, 2004. 44. Warren CM, Jordan MC, Roos KP, Krzesinski PR, Greaser ML. Titin isoform expression in normal and hypertensive myocardium. Cardiovasc Res 59: 86 –94, 2003. 44A.Warren CM, Krzesinski PR, Greaser ML. Vertical agarose gel electrophoresis and electroblotting of high molecular weight proteins. Electrophoresis 24: 1695–1702, 2003. 45. Wu Y, Bell SP, Trombitus K, Witt CC, Labeit S, LeWinter MM, Granzier H. Changes in titin isoform expression in pacing-induced cardiac failure give rise to increased passive muscle stiffness. Circulation 106: 1384 –1389, 2002. 295 • JULY 2008 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.32.247 on June 11, 2017 1. Anversa P, Leri A, Kajstura. Cardiac regeneration. J Am Coll Cardiol 47: 1769 –1776, 2006. 2. Appleton CP. Hemodynamic determinants of Doppler pulmonary venous flow velocity components: new insights from studies in lightly sedated normal dogs. J Am Coll Cardiol 30: 1562–1574, 1997. 3. Appleton CP, Hatle LK, Popp RL. Relation of transmitral flow velocity patterns to left ventricular diastolic function: new insights from a combined hemodynamic and Doppler echocardiographic study. J Am Coll Cardiol 12: 426 – 440, 1988. 4. Appleton CP, Firstenburg MS, Garcia MJ, Thomas JD. Diastolic function and dysfunction. Cardiol Clin 18: 513–546, 2000. 5. Bell SP, Nyland L, Tischler MD, McNabb M, Granzier G, LeWinter MM. Alterations in determinants of diastolic suction during pacing tachycardia. Circ Res 87: 235–240, 2000. 6. Brauch KM, Dhruv ND, Hanse EA, Andrews MT. Digital transcriptome analysis indicates adaptive mechanisms in the heart of a hibernating mammal. Physiol Genomics 23: 227–234, 2005. 7. Burlington RF, Darvish A. Low temperature performance of isolated working hearts from a hibernator and a nonhibernator. Physiol Zool l61: 387–395, 1998. 8. Caprette DR, Senturia JB. Isovolmetric performance of isolated ground squirrels and rat hearts at low temperature. Am J Physiol Regul Integr Comp Physiol 247: R722–R727, 1984. 9. Carzola O, Frieburg A, Helmes M, Centner T, McNabb M, Wu Y, Tombitas K, Labiet S, Granzier H. Differential expression of cardiac titin isoforms and modulation of cellular stiffness. Circ Res 86: 59 – 67, 2000. 10. Constanzo MR, Augustine S, Bourge R, Bristow M, OⴕConnell JB, Driscoll D, Rose E. Selection and treatment of candidates for heart transplantation. A statement for health professionals from the Committee on Heart Failure and Cardiac Transplantation of the Council on Clinical Cardiology, American Heart Association. Circulation 92: 3593–3612, 1995. 11. Eliceiri KW, Rueden C. Tools for visualizing multidimensional images from living specimens. Photochem Photobiol 81: 1116 –1122, 2005. 12. Folk GE Jr, Brewer MC, Sanders D. Cardiac physiology of polar bears in winter dens. Arctic 23: 130 –131, 1970. 13. Folk GE Jr, Hunt JM, Folk MA. Further evidence for hibernation of bears. In: Bears—Their Biology and Management, Papers and proceedings third international conference on bears—their biology and management, edited by Pelton MR, Lentfer JW, and Folk GE Jr. International Association of Bear Research and Management, 1974, p. 43– 47. 14. Fukuda N, Wu Y, Farman G, Irving TC, Granzier H. Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle. J Physiol 553: 147–154, 2003. 15. Garcia MJ, Firstenberg MS, Greenburg NL, Smedira N, Rodriguez L, Prior D, Thomas JD. Estimation of ventricular operating stiffness from Doppler early filling deceleration time in humans. Am J Physiol Heart Circ Physiol 280: H554 –H561, 2001. 16. Granzier H, Labeit S. Cardiac titin: an adjustable multi-functional spring. J Physiol 541: 335–342, 2002. 17. Granzier H, Labeit S. The giant protein titin: a major player in myocardial mechanics, signaling, and disease. Circ Res 94: 284 –295, 2004. 18. Hein S, Gaasch WH, Schaper J. Giant molecule titin and myocardial stiffness. Circulation 106: 1302–1304, 2002. 19. Hellyer P, Muir WW 3rd, Hubbell JA, Sally J. Cardiorespiratory effects of the intravenous administration of tiletamine-zolazepam to dogs. Vet Surg 18: 160 –165, 1989. 20. Hershey JD, Robbins CT, Nelson OL, Lin DC. Minimal seasonal alteration in the skeletal muscle of captive brown bears. Phyisol Biochem Zool 81: 138 –147, 2008. 21. Hissa R, Siekkinen J, Hohtola E, Saarela S, Hakala A, Puda J. Seasonal patterns in the physiology of the Eurpean brown bear (Ursus arctos arctos) in Finland. Comp Biochem Physiol 109A: 781–791, 1994. 22. Ishikawa J, Kario K, Matsui Y, Shibasaki S, Morninari M, Kaneda R, Hoshide S, Eguchi K, Hojo Y, Shimada K. Collagen metabolism in extracellular matrix may be involved in arterial stiffness in older hypertensive patients with left ventricular hypertrophy. Hypertens Res 28: 995–1001, 2005. 23. Kertesz NJ, Friedman RA, Colan SD, Walsh EP, Gajarski RJ, Gray PS, Shirley R, Geva T. Left ventricular mechanics and geometry in patients with congenital complete atrioventricular block. Circulation 96: 3430 –3435, 1997. 24. Lohuis TD, Harlow HJ, Beck TD, Iaizzo PA. Hibernating bears conserve muscle strength and maintain fatigue resistance. Physiol Biochem Zool 80: 257–269, 2007. H371