Development of dilated cardiomyopathy in Bmal1-deficient mice
Mellani Lefta,1,2 Kenneth S. Campbell,1,2 Han-Zhong Feng,3 Jian-Ping Jin,3 and Karyn A. Esser1,2
1Center for Muscle Biology, University of Kentucky, Lexington, Kentucky; 2Department of Physiology, College of Medicine,University of Kentucky, Lexington, Kentucky; and 3Department of Physiology, Wayne State University School of Medicine,Detroit, Michigan
Submitted 22 March 2012; accepted in final form 9 June 2012
Lefta M, Campbell KS, Feng H, Jin J, Esser KA. Developmentof dilated cardiomyopathy in Bmal1-deficient mice. Am J PhysiolHeart Circ Physiol 303: H475–H485, 2012. First published June 15,2012; doi:10.1152/ajpheart.00238.2012.—Circadian rhythms are ap-proximate 24-h oscillations in physiology and behavior. Circadianrhythm disruption has been associated with increased incidence ofhypertension, coronary artery disease, dyslipidemia, and other cardio-vascular pathologies in both humans and animal models. Mice lackingthe core circadian clock gene, brain and muscle aryl hydrocarbonreceptor nuclear translocator (ARNT)-like protein (Bmal1), are be-haviorally arrhythmic, die prematurely, and display a wide range oforgan pathologies. However, data are lacking on the role of Bmal1 onthe structural and functional integrity of cardiac muscle. In the presentstudy, we demonstrate that Bmal1�/� mice develop dilated cardio-myopathy with age, characterized by thinning of the myocardial walls,dilation of the left ventricle, and decreased cardiac performance.Shortly after birth the Bmal1�/� mice exhibit a transient increase inmyocardial weight, followed by regression and later onset of dilationand failure. Ex vivo working heart preparations revealed systolicventricular dysfunction at the onset of dilation and failure, precededby downregulation of both myosin heavy chain isoform mRNAs. Weobserved structural disorganization at the level of the sarcomere witha shift in titin isoform composition toward the stiffer N2B isoform.However, passive tension generation in single cardiomyocytes wasnot increased. Collectively, these findings suggest that the loss of thecircadian clock gene, Bmal1, gives rise to the development of anage-associated dilated cardiomyopathy, which is associated with shiftsin titin isoform composition, altered myosin heavy chain gene expres-sion, and disruption of sarcomere structure.
CIRCADIAN RHYTHMS are oscillations in animal physiology andbehavior that cycle with a period of �24 h. Underlyingcircadian behavior is the function of the molecular clock.Molecular clocks are intrinsic to each mammalian cell andgenerate cell autonomous and self-sustaining rhythms, whichare thought to prepare the organism for changes and/or stressesin the surrounding environment (26, 48, 54). Molecular clocksare composed of a series of interconnected transcriptional-translational positive and negative feedback loops, which gen-erate rhythmicity in gene expression, protein abundance, phys-iological processes, and animal behavior (38, 67). Brain andmuscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein (Bmal1) is one of the core molecular clock transcrip-tion factors and is a member of the basic helix loop helix-periodARNT single-minded (bHLH-PAS) family of transcription fac-tors (7). Upon dimerization with circadian locomotor output
cycles kaput (CLOCK), another member of the bHLH-PASfamily, it binds to E-boxes to regulate transcription of membersof the negative arm of the molecular clock, Period and Cryp-tochrome genes (21, 61), as well as other clock-controlledgenes that regulate an array of cellular processes (3). Bmal1 isunique among members of the core molecular clock, since itsloss in mice results in arrhythmic behavior as assessed byvoluntary wheel running (7). In addition to disrupted circadianbehavior, Bmal1�/� mice die young and are suggested to be amouse model of accelerated aging (34), with additional pathol-ogies including increased levels of liver and kidney functionenzymes (55), defective glucose homeostasis (51), skeletalmuscle weakness (2), increased sleep fragmentation (37), ar-thropathy (6), and infertility (1). In the cardiovascular system,Bmal1�/� mice lack the diurnal variation in heart rate andblood pressure and remain hypotensive throughout the circa-dian cycle (12). However, very little is known about thestructure and function of the Bmal1�/� hearts and whether anychanges occur in an age-associated manner.
A common pathology seen in aged humans and rodents isdilated cardiomyopathy (DCM) (11, 29, 56). DCM is a primarydisease of the myocardium and one of the leading causes ofcongestive heart failure, causing significant morbidity andpremature mortality (60). DCM is characterized by impairedmyocardial contractility [as assessed by a reduction in ejectionfraction, fractional shortening (FS), maximal left ventricularpressure, and rate of pressure development], dilation of the leftventricular chamber, and thinning of the ventricular walls (15,40, 53). The reduction in myocardial contractility is oftenassociated with changes in myosin heavy chain (MHC) isoformcomposition. Increased expression of MHC-� leads to a sig-nificant decrease in systolic function (58) and has been ob-served in canine DCM models and rat pressure overloadmodels (20, 28). A recent canine study of tachycardia-inducedDCM showed increased myocardial passive tension and thepossible involvement of the striated muscle protein titin (65).Two titin isoforms, differing in stiffness properties, are coex-pressed in the heart and compositional changes occur in re-sponse to different physiological demands imposed on themyocardium (18, 23). Titin plays an important role in myofil-ament repair and turnover (25), a process that could be dis-rupted in circadian dysfunction (5).
In this study, we evaluated cardiac performance starting at 4through 36 wk of age using noninvasive echocardiography andwe show the development of an age-associated DCM pheno-type in the Bmal1�/� mice. Structural analysis using electronmicroscopy indicates a disruption of sarcomere architecture inthe Bmal1�/� hearts. This is associated with downregulation ofboth MHC mRNA isoforms at ages before the development ofDCM, but not MHC isoform protein levels. Biochemical anal-ysis of whole heart extracts reveals a shift in titin isoforms, but
Address for reprint requests and other correspondence: K. Esser, Ctr. forMuscle Biology, Dept. of Physiology, Chandler Medical Ctr., MS 508, Lex-ington, KY, 40536 (e-mail: [email protected]).
Am J Physiol Heart Circ Physiol 303: H475–H485, 2012.First published June 15, 2012; doi:10.1152/ajpheart.00238.2012.
we did not detect a change in passive stiffness at the level ofthe single cardiomyocyte. Systolic performance is decreasedbefore the overt development of DCM in the Bmal1�/� mice,as shown by ex vivo functional measurements of isolatedworking hearts. Together, these findings suggest that loss ofBmal1 results in an age-associated pathology in the heart thatshares some, but not all, characteristics with DCM.
MATERIALS AND METHODS
All animal procedures were conducted in compliance with the guide-lines of the Association for Assessment and Accreditation of LaboratoryAnimal Care and were approved by the Institutional Animal Care andUse Committees at University of Kentucky and Wayne State University.
Animals. The germline Bmal1�/� mice were previously backcrossedover 10 generations to the C57BL6 background (2). Both male andfemale mice are infertile (1), so the Bmal1�/� and wild-type mice for thisstudy were littermates from heterozygote (Bmal1�/�) breeding. Geno-types were determined as previously described (7). Mice were housedfour per cage and kept on a 14-h:10-h light-dark schedule with ad libitumaccess to food and water.
Echocardiograms. Mice were placed on a heated platform set at37°C and anesthetized with isoflurane gas, and transthoracic measure-ments were taken using a VisualSonics Vevo 660 with a RMV 70730-MHz probe. M-mode images were acquired from the parasternalshort-axis view at the papillary muscle level. Data analysis wasperformed with the use of the Vevo 660 analytic software.
Cardiomyocyte isolation. Cardiomyocytes were isolated as previ-ously described (45a, 68). The composition of all buffers is identicalto those of O’Connell et al. (45a), except that trypsin was not includedin the digestion buffer. Briefly, mice were euthanized by cervicaldislocation, the heart was carefully excised, and the aorta was cannu-lated. The heart was hung in a Langendorff apparatus and perfusedretrograde with aerated (95% O2-5% CO2, 37°C) perfusion buffer,followed by perfusion with digestion buffer until the heart was paleand swollen. Following digestion, the ventricles were minced, resus-pended in digestion buffer, and triturated to create a cell suspension.An equal amount of Stop-1 solution was added and the cardiomyo-cytes were allowed to settle into a pellet. The pellet was resuspendedin Stop-2 solution, and the isolation was considered successful whenabout 65% of the cells were rod shaped. Isolated cardiomyocytes wereused for assessment of cell size or for passive tension measurements.
Cardiomyocyte size determination. Cardiomyocytes in Stop-2 so-lution were treated with Hoechst dye (1:1,000) for 5 min. A hundredimages were taken from each heart at �100 magnification using aNikon Eclipse E600 light microscope, averaging 275 cells per heart.Cardiomyocyte area was measured manually using National Institutesof Health (NIH) ImageJ software (10). Cardiomyocytes were binnedaccording to their cell area in bins of 500 �m2, and histograms withthe percentage of cardiomyocytes in each bin were generated usingGraphPad Prism (43).
Histological sections. At 36 wk of age, mice were euthanized bycervical dislocation; the heart was excised and placed in a dish containingwarm phosphate-buffered saline until it stopped beating, placed in 4%paraformaldehyde (24 h at 4°C), and then in 70% ethanol. The fixedhearts were embedded in paraffin, and 5-�m sections were taken startingat the papillary muscle level. Sections were stained with Masson’sTrichrome (Sigma-Aldrich, St. Louis, MO). Low-magnification images(�20) were taken using a Nikon Eclipse E600 light microscope. Leftventricular wall thickness and area were obtained manually using NIHImageJ software (10).
Electron microscopy. Mice were anesthetized with ketamine-xyla-zine, the chest cavity was opened, and the heart was exposed.Perfusion through the left ventricle was started first with phosphate-buffered saline (pH 7.4), followed by cold 2% paraformaldehyde-4%glutaraldehyde in 0.1 M sodium cocadylate buffer (pH 7.4) and 130mM NaCl. The perfusion-fixed hearts were then taken to the electron
microscopy core facility at the University of Kentucky for processingand sectioning (14). Images were obtained using a Philips Biotwin 12transmission electron microscope.
Real-time PCR. Total RNA was extracted from 4-wk-old wild-typeand Bmal1�/� hearts using TRIzol (Invitrogen). Isolated RNA wasquantified using the Nano Drop 2000 spectrophotometer (ThermoScientific). First-strand cDNA synthesis was done using SuperScriptIII first-strand synthesis supermix kit (Invitrogen). Real-time PCR wasperformed using TaqMan probe-based chemistry (Applied Biosys-tems) and conducted in an ABI 7700 sequence detector. Probes andprimers for Gapdh, Myh6, and Myh7 were purchased from AppliedBiosystems. After normalization to Gapdh [change in cycle threshold(�Ct)], gene expression was reported relative to the average expres-sion for that gene in the wild-type group at 4 wk of age (��Ct). Foldchange relative to control was calculated as 2���Ct.
Sample preparation for MHC gels. MHC protein expression wasperformed on ventricular tissue from 4-, 12-, and 36-wk-old wild-typeand Bmal1�/� mice. Control tissue for MHC-� and MHC-� was fromneonatal hearts. Frozen ventricular tissue was homogenized using ninevolumes of homogenizing buffer/mg tissue, consisting of (in mM) 250sucrose, 25 NaCl, and 20 Tris (pH 7.4), modified from Talmadge andRoy (57). The homogenate was spun at 20,000 g for 30 min at 4°C.The pellet, which was enriched in myofibrils, was resuspended inhomogenizing buffer and protein concentration was determined usinga Bradford assay. Sample buffer (3�), consisting of 1.15 M Tris (pH6.8), 6% SDS, 75 mM DTT, 0.06% bromophenol blue, and 40%glycerol, was added to a final concentration of 1�. The samples wereheated at 100°C for 2 min, and 0.8 �g/lane was immediately loadedon gels (57).
MHC electrophoresis and isoform determination. Gel compositionand preparation were identical to Talmadge and Roy (57). The upper andlower chamber buffer composition was the same as in Reiser and Kline(49). The gels were run at 70-V constant voltage for 38 h at 4°C[modified from (57)]. MHC isoforms were visualized with a silver stainplus kit (Bio-Rad, Philadelphia, PA) and scanned using an Epson Per-fection V500 photo scanner. Total MHC and the percentage of MHC-�and MHC-� were determined using NIH ImageJ software (10).
Sample preparation for titin gels. Frozen ventricular tissue wasplaced in sample buffer [described in Warren et al. (63), excludingbromophenol blue, buffer-to-tissue ratio 60:1 (vol/wt)]. Protein con-centration of the homogenate was determined using the RC-DCprotein assay (Bio-Rad). Samples were heated at 60°C for 10 min;glycerol and bromophenol blue were added to 30% (vol/vol) and 0.1%(vol/vol) final concentration, respectively. Following 10 min centrif-ugation at 13,000 g, the supernatant was aliquoted and loaded on gelor stored at �80°C.
Titin electrophoresis and isoform determination. A Bio-Rad Pro-tean II xi XL vertical electrophoresis unit was used. The gel size was20 cm � 20 cm � 1.5 mm. The composition of the acrylamide plug,the agarose gel, and the upper and lower chamber buffers were thesame as in Warren et al. (63). The gels were run at 4°C for a total of12 h, starting at 80 V and increasing the voltage by 30 V every 100min. After electrophoresis, the gels were stained with Sypro Ruby(Invitrogen, Eugene, OR) and scanned using a Typhoon scanneroperating in fluorescence mode at 450 nm. Scanned images wereanalyzed using Image Quant software.
Ex vivo working heart preparation and functional measurements.Cardiac function was measured in isolated working heart preparationsof 18-wk-old male wild-type and Bmal1�/� mice. As describedpreviously (16), the mice were heparinized and anesthetized beforethe heart was rapidly isolated. A modified 18-gauge, 6-mm-long,thin-walled needle was used as the aortic cannula. After retrogradeperfusion was established, a modified 16-gauge needle was used tocannulate the pulmonary vein for antegrade perfusion through the leftatrium. A 30-gauge needle was used to puncture the left ventricle fromthe apex to make a path for the insertion of a 1.2-Fr pressure-volume
H476 DILATED CARDIOMYOPATHY IN Bmal1-DEFICIENT MICE
catheter (calibrated for pressure and volume at 37°C, model 898B,Scisense, London, Ontario, Canada).
Aortic pressure was measured using an MLT844 pressure trans-ducer (Capto, Horten, Norway) that was placed at heart level andconnected to the aortic cannula. A 0.5-ml air bubble was placed in thecompliance chamber to mimic in vivo aortic compliance and abeveled polyethylene-50 tubing was used to cannulate the pulmonaryartery to collect coronary effluent. In all experiments, the hearts wereperfused with modified Krebs-Henseleit buffer aerated with 95%O2-5% CO2 at 37°C without recycling to exclude the effects ofmetabolic products and hormones. The buffer contents were as fol-lows: (in mM) 118 NaCl, 4.7 KCl, 2.25 CaCl2, 2.25 MgSO4, 1.2KH2PO4, 0.32 EGTA, 25 NaHCO3, 15 D-glucose, and 2 sodiumpyruvate (pH 7.4, adjusted at 37°C). Heart rate was controlled at 480beats/min by supraventricular pacing using an isolated stimulator(A365, World Precision Instrument) with two microplatinum elec-trodes attached to the right atrium. Cardiac output was measured bythe actual aortic and pulmonary artery flows recorded in real time bycalibrated drop counting using a pair of electrodes feeding to aPowerLab/16 SP digital data archiving system (AD Instruments).Baseline function of the isolated working hearts was measured at apreload of 10 mmHg and afterload of 55 mmHg for the maximum leftventricular pressure, the maximum rate of left ventricular pressuredevelopment (dP/dt), and left ventricular volume. Left ventricularstroke volume (�l/mg heart tissue) was calculated from the sum ofaortic flow and coronary effluents, normalized to heart rate.
Single cardiomyocyte mechanical measurements. Passive tensionwas measured in single cardiomyocytes isolated from wild-type andBmal1�/� hearts, using a method described by Warren et al. (62) witha few modifications. The composition of relaxing and pCa9 solutionswas identical to Ferreira et al. (17). Cardiomyocytes were resuspendedin relaxing solution and were chemically permeabilized in relaxingsolution containing 0.5% (vol/vol) Triton X-100 (Thermo Scientific,Rockford, IL) for 6 min at room temperature. Cardiomyocytes werewashed twice in relaxing solution and stored on ice for up to 12 h.Cardiomyocytes were attached between stainless steel insect pinsextending from a motor (308B, Aurora Scientific, Ontario, Canada)and a force transducer (406, Aurora Scientific) using silicone adhe-sive. Once attached, the cardiomyocyte was lifted and placed in a wellcontaining pCa9 solution at 15°C. This apparatus was placed on thestage of a Nikon TE2000-U-inverted microscope with the capacity forvideo capturing. Experiments were performed using SLControl soft-ware (8). Each cardiomyocyte was stretched to double its restinglength at a rate of 0.1 l0/s, and then shortened to 50% l0 before beingreturned to its resting length. The SLControl software generated tracesof passive tension and fiber length over the stretch time. A video of thestretch was captured for subsequent analysis. The SLControl data andthe video file were transported to a MatLab program written by K. S.Campbell, which extracted the passive tension values from theSLControl data and plotted them against sarcomere length calculatedfrom individual frames from the video file. We used 20–24 prepara-tions from four mice from each age and genotype group. Experimentalresults from each group were binned according to their sarcomerelengths in bins of 0.05 �m, and a two-way ANOVA with a post hocBonferroni test was run to assess significance. Additionally, passivetension traces from each cardiomyocyte were analyzed with an expo-nential equation: passive tension exp[k·(SL � SL0)] (19), where SLis the sarcomere length, k is the exponent of stiffness, and SL0 is thesarcomere length at zero load.
Statistical analysis. Results from each group (Bmal1�/� vs. wild-type) at each time point are reported as means SE. Two-wayANOVA was performed to determine whether a significant interactionexisted between factors for each dependent variable under consider-ation. If a significant interaction was detected, Bonferroni post hoccomparisons were performed to identify the source of significance,with P � 0.05.
RESULTS
Germline Bmal1�/� mice develop age-associated DCM. Echocar-diogram data showed that at 4 wk of age, the germline Bmal1�/�
mice had bigger hearts compared with wild-type littermates, asdemonstrated by a 15% increase in the estimated left ventricularweight (LVW) (129 13.79 vs. 110 6.57 mg) (Fig. 1A) and a43% increase in ratio of LVW to body weight (BW) (8.46 1.14vs. 5.9 0.4 mg/g, P � 0.001) (Fig. 1B). We measured wet LVWand heart weight (HW) in a separate cohort of mice to comple-ment echocardiographic estimates and to show that LVW wasincreased by 19% in the Bmal1�/� mice, but this increase was notstatistically significant (59.47 7.40 vs. 49.98 8.31 mg) (Fig.1H). However, because of the smaller body size in Bmal1�/�
mice, wet LVW-to-BW ratio was significantly increased by 56%(4.74 0.50 vs. 3.04 0.64 mg/g, P � 0.05) as was theHW-to-BW ratio (6.26 0.29 vs. 5.26 0.37 mg/g) (data notshown). At this age, FS was preserved (43.65 3.14 vs. 41.05 1.96%) (Fig. 1C), the interventricular septum (IVS) was slightly,but not significantly, thicker (1.62 0.08 vs. 1.54 0.08 mm)(Fig. 1D), and no changes were detected in the left ventricularinternal diameter (LVID) (1.67 0.08 vs. 1.68 0.07 mm) (Fig.1E) or left ventricular posterior wall (LVPW) thickness (Fig. 1F).To evaluate whether the increase in LVW-to-BW ratio wasassociated with hypertrophy of cardiomyocytes, we isolated car-diomyocytes and measured cell area. We found that Bmal1�/�
hearts have a greater percentage of smaller cardiomyocytes com-pared with wild-type hearts (Fig. 2A). The percentage of cardio-myocytes smaller than 2,500 �m2 is significantly higher in theBmal1�/� hearts (56.41 3.23 vs. 38 4.22 �m2, P � 0.01),whereas the percentage of larger cardiomyocytes is significantlydecreased (43.59 3.23 vs. 62 4.22 �m2, P � 0.01) (Fig. 2B).The average cardiomyocyte area is 19% smaller in the Bmal1�/�
hearts (2,538 470.88 vs. 3,152 796 �m2) (Fig. 2C).This increase in LVW-to-BW seen in the 4-wk-old Bmal1�/�
mice began to regress by 8 wk of age and declined to wild-typelevels by 12 wk of age as assessed by echocardiography (4.67 0.28 vs. 4.48 0.25 mg/g) (Fig. 1B) and wet weight measure-ments (3.38 0.18 vs. 3.56 0.05 mg/g) (data not shown).Weeks 8–16 were characterized by a regression of LVW-to-BWratio and myocardial wall thickness, whereas FS continued to bepreserved (Fig. 1C). The IVS and LVPW started to get thinner inthe Bmal1�/� mice compared with wild-type mice, but thesedifferences did not reach statistical significance by 16 wk of age(IVS, 1.36 0.07 vs. 1.53 0.03 mm; and LVPW, 1.13 0.04vs. 1.35 0.07 mm) (Fig. 1, D and F). Additionally, the LVIDwas similar in wild-type and Bmal1�/� hearts (Fig. 1E).
Weeks 20–36 were characterized by a significant decline inFS, indicating systolic dysfunction in the Bmal1�/� mice. By36 wk of age, FS was 24.3% smaller in the Bmal1�/� micecompared with wild-type mice (24.6 1.16 vs. 32.5 1.06%,P � 0.01) (Fig. 1C). During this time period, the IVS and theLVPW continued to thin and by 36 wk of age were on average26.67 and 24.8% smaller in the Bmal1�/� mice (IVS, 1.10 0.05 vs. 1.50 0.05 mm, P � 0.001; and LVID, 0.97 0.05vs. 1.29 0.04 mm, P � 0.05) (Fig. 1, D and F). The LVIDgot progressively larger in the Bmal1�/� mice during this time,becoming 17.12% larger in the Bmal1�/� mice compared withwild-type by 36 wk of age (3.01 0.10 vs. 2.57 0.11 mm,P � 0.05) (Fig. 1E). LVW declined during this time and by 36wk of age was 22.33% smaller in the Bmal1�/� mice (88.54
H477DILATED CARDIOMYOPATHY IN Bmal1-DEFICIENT MICE
3.16 vs. 114.0 6.65 mg) (Fig. 1A). This was during a periodof time in which body weight continued to decline (Fig. 1G),thus LVW-to-BW ratio remained unchanged as estimated byechocardiography (3.96 0.20 vs. 3.68 0.24 mg/g) (Fig.1B) and wet weight measurements (3.46 0.28 vs. 3.48 0.28 mg/g) (data not shown). It is also worth noting that bodyweight (19.90 1.40 vs. 31.11 1.40 g, P � 0.001), wetLVW (66.67 2.91 vs. 107.74 9.13 mg, P � 0.001), andwet HW (113.86 3.92 vs. 157.06 11.62 mg, P � 0.01)were significantly smaller in the Bmal1�/� mice at 36 wk ofage (Fig. 1, G–I).
At 36 wk of age, we evaluated histological cross sections ofthe heart at the level of the papillary muscles, and this analysissupported the echocardiography data. Representative imagesare shown in Fig. 3, A and B. We found that the area occupiedby myocardium was decreased by 21% in the Bmal1�/� mice(5.54 0.21 vs. 7.05 0.57 mm2, P � 0.05) (Fig. 3C). IVSthickness was decreased by 20% (0.68 0.03 vs. 0.85 0.07
mm, P � 0.05), and LVPW thickness was decreased by 31%(0.70 0.03 vs. 1.02 0.09 mm, P � 0.01) in the Bmal1�/�
mice compared with wild-type controls (Fig. 3D). The leftventricular cavity area was slightly increased (3.13 0.20 vs.2.75 0.16 mm2), but this difference did not reach statisticalsignificance (Fig. 3C).
Bmal1�/� myocardium exhibits disorganized sarcomeres. Toassess whether the cardiac functional changes were associatedwith disruptions in sarcomere architecture, we obtained electronmicrographs from wild-type and Bmal1�/� myocardial sec-tions at 14 wk of age. An evaluation of electron micrographsfound that some regions had normal sarcomere architecture inthe Bmal1�/� myocardium, whereas other areas exhibitedsarcomere disorganization, with diffuse M lines, A bands, andZ disks. We found regions where the distinction between A andI bands was diminished in the Bmal1�/� myocardium, and thiswas not seen in any of the images from the wild-type hearts(Fig. 4, A and B).
Fig. 1. Age-associated dilated cardiomyopathy development in germline brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein-1knockout (Bmal1�/�) mice. Graphs show longitudinal echocardiographic changes in left ventricular (LV) mass (A), LV weight (LVW) normalized to body weight(BW) (B), fractional shortening (C), systolic interventricular septum thickness (IVS; D) systolic LV internal diameter (LVID; E), systolic LV posterior wallthickness (LVPW; F), and BW (G). Data are means SE; n 7. The numbers in the white boxes denote the stage of cardiac pathology observed in that agerange. Summarized data for wet LVW (H) and wet heart weight (I) from wild-type (WT) and Bmal1�/� mice at each representative age are shown. Values aremeans SE; n 4 (4 wk and 12 wk), 8 (18 wk), and 7 (36 wk). *P � 0.05; **P � 0.01; ***P � 0.001.
H478 DILATED CARDIOMYOPATHY IN Bmal1-DEFICIENT MICE
mRNA, but not protein, levels of MHC isoforms were down-regulated in the Bmal1�/� myocardium at 4 and 12 wk of age. Wemeasured mRNA expression of MHC-� and MHC-� in theBmal1�/� and wild-type hearts at all three ages (Fig. 5, A and B).
MHC-� mRNA levels were significantly decreased by 31 and32% at 4 and 12 wk, respectively, and were unchanged at 36wk (Fig. 5A). MHC-� mRNA expression was significantlylower by 57 and 59% at 4 and 12 wk, respectively, but was not
Fig. 2. Bmal1�/� cardiomyocytes are smallerthan WT cardiomyocytes at 4 wk of age.Average cardiomyocyte area from WT andBmal1�/� mice at 4 wk of age is represented,showing that cardiomyocytes from Bmal1�/�
hearts are smaller (C). Cardiomyocytes werebinned according to their size in 500-�m2
bins, and the percentage of cardiomyocytes ineach bin was calculated. There is a shift towardsmaller cardiomyocytes in the Bmal1�/� mice(A). The percentage of cardiomyocytes smallerthan 2,500 �m2 was increased in the Bmal1�/�
hearts, whereas the percentage of larger cardio-myocytes (�2,500 �m2) was decreased (B).n 880 cells from 4 mice (WT) and 1,330 cellsfrom 4 mice (Bmal1�/�). *P � 0.05; **P �0.01; ***P � 0.001.
Fig. 3. Thinning of the myocardial walls and dilation ofthe ventricular cavity in 36-wk-old Bmal1�/� mice.Representative Masson’s trichrome-stained myocardialsections at the level of the papillary muscles, showenlargement of the ventricular cavity and wall thinningin the 36-wk-old Bmal1�/� myocardium (B) comparedwith an age- and sex-matched WT control myocardium(A). Graphs of combined data show a decrease in thearea of myocardium (C) and the thickness of both theIVS and LVPW (D). LVC, LV cavity. n 5 WT and 10Bmal1�/� mice. *P � 0.05; **P � 0.01.
H479DILATED CARDIOMYOPATHY IN Bmal1-DEFICIENT MICE
statistically different at 36 wk (Fig. 5B). MHC-� mRNAincreased with age in both the wild-type and Bmal1�/� groups.However, we found no difference in MHC isoform composi-tion or total MHC content at the protein level between wild-type and Bmal1�/� hearts (Fig. 5, C and D).
Bmal1�/� mice show increase of N2B and decrease of N2BAtitin isoforms. Total titin, reflecting the sum of the N2BA, N2B,and T2 bands, and normalized to MHC and was not differentbetween wild-type and Bmal1�/� hearts at any age (Fig. 6B).Additionally, the percentage of intact titin, calculated as thesum of N2BA and N2B bands over the sum of N2BA, N2B,and T2, was not affected by genotype and ranged between 83and 90% in the wild-type hearts and 86 and 91% in theBmal1�/� hearts (Fig. 6C). The T2 band is a degradativeproduct of titin and is a measure of both in vivo degradation aswell as degradation that could have occurred during processingof the samples. The levels of total titin normalized to MHCwere similar in Bmal1�/� and wild-type hearts during stage 1,increased LVW-to-BW ratio (0.24 0.07 vs. 0.23 0.019);
stage 2, regression (0.24 0.027 vs. 0.27 0.017); and stage3, dilation and failure (0.20 0.014 vs. 0.22 0.011),suggesting that the cardiac pathology is not a result of changesin total titin protein levels.
However, we found that the percentage of the compliant N2BAisoform, calculated as N2BA/(N2BA � N2B), was lower in theBmal1�/� left ventricles, especially at 4 wk (14.24 1.73 vs.19.79 0.82%, P � 0.05) and 36 wk of age (12.73 0.76 vs.18.32 0.90%, P � 0.01) (Fig. 6D). This was at the expense of thestiffer N2B titin isoform, which was in turn increased in Bmal1�/�
hearts compared with age-matched wild-type controls (4 wk,85.76 1.7 vs. 80.21 0.82%, P � 0.05; 12 wk, 85.34 0.29vs. 83.52 1.64%; and 36 wk, 87.27 0.76 vs. 82.22 0.94%,P � 0.01) (Fig. 6E). The N2BA-to-N2B ratio, a well-acceptedmeasure of myocardial stiffness, was decreased in the Bmal1�/�
hearts (Fig. 6F) during stage 1 when LVW to BW is increased(0.17 0.02 vs. 0.25 0.01, P � 0.05) and stage 3 when theheart is dilated and failing (0.15 0.01 vs. 0.23 0.01, P �0.01) but not during stage 2 (0.17 0.004 vs. 0.20 0.02)
Fig. 4. Bmal1�/� myocardium exhibits disor-ganized sarcomeres. Representative electronmicrographs from WT (A) and Bmal1�/� (B)hearts are shown. Bmal1�/� sarcomeres haveless defined A bands and I bands and diffuse Mlines and Z disks compared with sarcomeresfrom WT hearts.
Fig. 5. Myosin heavy chain (MHC) RNA is downregulated at 4 and 12 wk of age, whereas protein content and isoform composition is not different in theBmal1�/� myocardium. Real-time PCR data show downregulation of both MHC-� (A) and MHC-� (B) in Bmal1�/� hearts at 4 and 12 wk of age, but not at36 wk. Representative SDS-PAGE for MHC isoform separation show that the left ventricles of WT and Bmal1�/� mice contain only the MHC-� isoform (D).Total MHC is expressed relative to the neonatal heart sample to normalize for gel to gel differences (C). For the RT-PCR experiment, n 7 (4 wk), 4 (12 wk),and 6 (36 wk). *P � 0.05; ***P � 0.001. For the MHC gels, n 4 mice for each age and genotype.
H480 DILATED CARDIOMYOPATHY IN Bmal1-DEFICIENT MICE
when the wild-type and Bmal1�/� hearts were functionallysimilar.
Bmal1�/� hearts exhibit compromised systolic function in exvivo preparations. To complement the echocardiography data,we measured cardiac function in isolated working hearts fromwild-type and Bmal1�/� mice at 18 wk of age, the beginning ofstage 3. As seen in Fig. 7, hearts from Bmal1�/� mice exhibitedsignificant depression in left ventricular systolic function as dem-onstrated by a 9.7% decrease in maximal left ventricular pressure(Fig. 7C), a 22.3% decrease in the rate of contraction (dP/dt) (Fig.7D), and a subsequent 24.7% decrease in stroke volume (Fig. 7B).At this time point, the heart was larger as shown by the increasein HW-to-BW ratio (Fig. 7A), whereas the rate of relaxation(�dP/dt) was not affected (Fig. 7D).
Passive tension is not different in Bmal1�/� cardiomyocytescompared with wild-type controls. We found no difference inpassive tension between Bmal1�/� and wild-type cardiomyo-cytes at any age (at sarcomere length of 2.45 �m, 4 wk:13.63 0.75 vs. 11.54 1.28 kN/m2; 12 wk: 10.16 0.72 vs.12.54 1.25 kN/m2; 36 wk: 8.78 0.65 vs. 8.78 0.32kN/m2) (Fig. 8). Additionally, the exponential values, k andSL0 were not different between Bmal1�/� and wild-type car-diomyocytes at any age (Fig. 8).
DISCUSSION
Data presented in this study demonstrate that germlineBmal1�/� mice develop age-associated DCM associated withmyofilament disorganization that is preceded by changes intitin isoform composition and downregulation of MHC tran-script levels. Whereas there is a rich body of evidence linking
circadian rhythm dysfunction to the development of cardiovas-cular pathologies (27, 32, 33, 42), data are lacking on the roleof Bmal1 and/or circadian rhythms on the structure and func-tion of the heart with age.
We followed a cohort of Bmal1�/� and wild-type mice withechocardiograms starting at 4 wk of age until they reached 36wk of age. We chose 36 wk as our end point because it hasbeen previously reported that Bmal1�/� mice have an averagelifespan of 37 12 wk (34). We identified three distinct stagesin the progression of the cardiac pathology in the Bmal1�/�
mice, with stage 1 at 4 wk, characterized by larger hearts in theBmal1�/� mice, whereas FS was not affected. We referred tothis first stage as a transitory increase in LVW-to-BW ratio,because it was not sustained past 4 wk of age. This indicatesthat the germline Bmal1�/� mice are different from wild-typemice beginning shortly after birth and have bigger hearts fortheir body weight.
We defined the changes in the heart between 8 and 16 wk ofage as stage 2, and this was characterized by regression ofLVW-to-BW ratio with preservation of FS. During stage 3(20–36 wk), there was significant thinning of the ventricularwalls and enlargement of the LVID. These morphologicalchanges were associated with a progressive decline in FS toaround 55% of the initial value and a significant depression incardiac systolic function as shown by decreased stroke volume,maximum left ventricular pressure, and dP/dt in the Bmal1�/�
hearts at the beginning of stage 3. These findings indicate thatloss of Bmal1 contributes to the development of featuresconsistent with DCM. The Bmal1�/� mice have been consid-ered a model of accelerated aging (34), and as such the
Fig. 6. Bmal1�/� mice (BM) show increase of N2B anddecrease of N2BA titin isoforms. Representative SDS-vertical agarose gel electrophoresis for titin protein isshown (A). Total titin-to-MHC ratio (B) and the percentageof intact titin (C) were not different between WT andBmal1�/� mice at any age. The percentage of the compli-ant N2BA isoform is decreased in the Bmal1�/� micecompared with age-matched controls (D), and this is as-sociated with an increase in the percentage of the stifferN2B isoform (E). These changes result in a decrease in theN2BA-to-N2B ratio in the 4- and 36-wk-old Bmal1�/�
mice compared with WT controls (F). n 4. *P � 0.05;**P � 0.01.
H481DILATED CARDIOMYOPATHY IN Bmal1-DEFICIENT MICE
pathologies observed here could be the result of acceleratedsystemic aging.
One feature we found early in the Bmal1�/� mouse is anenlarged heart seen with echocardiography and LVW-to-BWanalysis. However, upon analysis of cardiomyocyte size wefound that the Bmal1�/� hearts at 4 wk of age had largerpercentages of smaller cardiomyocytes and average cardiomy-ocyte size was reduced by 19%. This suggests that there weremore cells, possibly hyperplasia of cardiomyocytes or othercell types to support the larger heart size. Since cardiomyocytehyperplasia occurs mainly during embryonic development andup to 1 wk postnatally (46), our data suggest a possible role ofBmal1 in regulating the timing of cardiac development andgrowth. The link between loss of Bmal1 in the heart and heartsize in our studies is consistent with the findings by Durganet al. (13), in which cardiac specific loss of Bmal1 wasassociated with increased biventricular weight-to-BW ratio andincreased expression of the hypertrophic marker, mcip1. How-ever, the hypertrophy we observed in the germline Bmal1�/�
mouse occurs much earlier in life, at 4 wk of age, than thatobserved in the cardiac specific Bmal1�/� mouse, and the hearthypertrophy we detect is transient. Whether the increased sizein the Durgan study is due to hyperplasia and whether thehypertrophy persists are not known. Thus there is still much tolearn about the interaction among Bmal1, cardiomyocyte num-ber, and size in the heart.
Much of the data in this study comes from longitudinalechocardiography studies, and, like others, we found that heartrate in the Bmal1�/� mice was significantly lower compared
with wild-type mice at all ages (means SD across all timepoints, 406 49 vs. 482 41 beats/min). To minimize anypotential problems with interpretation, we attempted to matchheart rate for echocardiography through an adjustment ofisoflurane concentration; however, Bmal1�/� mice were moresensitive to isoflurane and displayed a quicker drop in heartrate. Previous studies have shown that cardiac parametersmeasured through M-mode echocardiography were stable overa 20-min time span and a range of heart rates from 450–550beats/min (50), whereas others have found increased LVW andLVID, decreased FS, and no effect on IVS and LVPW thick-ness when heart rate decreased by 120 beats/min (64). In thecurrent study, we see a 76 beats/min decrease in heart rate inthe Bmal1�/� mice, whereas our wild-type values for heart rateand FS are consistent with previously published work (22, 64).The findings that IVS and LVPW thickness and LVW aredecreased in our Bmal1�/� mice cannot be explained by thedecrease in heart rate and suggest that the changes seen in theBmal1�/� hearts are due to the pathology they develop and notan artifact of their decreased heart rate.
One characteristic of DCM is impairment in force genera-tion. The observed decline in the rate of contraction in theBmal1�/� hearts ex vivo suggested the involvement of MHC,since a shift to the slower MHC-� isoform is both correlatedwith decreased dP/dt (41) and commonly associated with DCM(20). It has been shown that a shift toward MHC-� occurs incanine models of DCM and a rat model of pressure overload(20, 28). Additionally, a forced expression of MHC-� protectsagainst cardiomyopathy in the rabbit left ventricle (31). Wefound that levels of MHC-� mRNA were not increased in theBmal1�/� hearts at any of the ages in which significantfunctional changes were detected with ex vivo analysis orechocardiography. This indicates that the pathology observedin the Bmal1�/� hearts is not associated with MHC isoformchanges and does not directly mimic models of DCM. What wedid find is that expression of MHC-� and MHC-� mRNAswere downregulated in the Bmal1�/� hearts at 4 and 12 wk.This is consistent with previous reports from skeletal muscle ofclock-compromised mice, showing downregulation of the mR-NAs for many sarcomeric and structural genes (39). We weresurprised to observe decreases in both MHC isoform mRNAlevels with no differences in protein levels at the 4 and 12 wkages. This suggests that in the early ages, loss of Bmal1 leadsto altered MHC gene expression but posttranscriptional, poten-tially protein turnover, mechanisms are in place to maintainnormal MHC protein levels. If MHC protein turnover is al-tered, it is possible that the MHC protein present in theBmal1�/� hearts may be modified and thus exhibit alteredfunction which could contribute to the observed decline inforce development and the rate of contraction in the Bmal1�/�
hearts. Additionally, a decrease in calcium release; mutationsin myosin light chain, actin, and troponin T; and alteredphosophorylation status of troponin I are common findings inDCM and affect cross-bridge cycle dynamics, actin-myosinATPase activity, and excitation-contraction coupling leading todecreased force generation (30, 52, 66). Much remains to bedone to understand the relationship between Bmal1 and cardi-omyocyte specific gene expression and maintenance of me-chanical function in the heart.
Titin plays a critical role in maintaining sarcomere structure.Our total titin/MHC values were similar to previously pub-
Fig. 7. Decreased systolic heart function in Bmal1�/� mice. Wet heart weight(in mg) normalized to BW (in g) was significantly higher in Bmal1�/� micecompared with the WT group (A). The functional measurements of ex vivoworking heart at preload of 10 mmHg and afterload of 55 mmHg with heartrate paced at 480 beats/min detected that Bmal1�/� hearts had deceased strokevolume (B), lower maximum left ventricular pressure (LVPmax; C), and slowersystolic velocity (�dP/dt; D) compared with the WT group. No changes werefound in diastolic velocity (�dP/dt; D) or end-diastolic left ventricular pres-sure (LVPmin; C). Values are means SE; n 4 WT and 5 Bmal1�/� mice.*P � 0.05 and ***P � 0.001 compared with WT in Student’s t-test.
H482 DILATED CARDIOMYOPATHY IN Bmal1-DEFICIENT MICE
lished work (9, 44, 65) and were not different between wild-type and Bmal1�/� mice at any age, suggesting that changes intotal titin are not mediators of the structural or functionalcardiac pathology observed in the Bmal1�/� mice. In themyocardium, two titin isoforms are expressed, the larger andmore compliant N2BA and the smaller, stiffer N2B (23). TheN2BA-to-N2B ratio, a measure of cardiac compliance, in ourwild-type mice was consistent with previously reported valuesfor mouse myocardium (45, 47). The N2BA-to-N2B ratiosignificantly decreased in the Bmal1�/� mice, specifically at 4wk of age when mice had increased LVW-to-BW ratio and at36 wk of age when mice showed most symptoms of DCM.These data are consistent with the canine study by Wu andcolleagues (65), in which rapid-pacing induced DCM resultedin a decrease in the N2BA-to-N2B ratio and a concomitantincrease in titin based passive tension. These data suggest thatloss of Bmal1 shifts titin isoform composition toward the stifferisoform and this shift potentially mediates the pathologies seenin the hearts of the Bmal1-deficient mice.
Titin-based passive force constitutes the majority of the totalpassive tension in cardiac muscle, and previous studies haveshown a correlation between changes in titin isoform compo-sition and myocardial stiffness (24). Although we found adecrease in the N2BA-to-N2B ratio, we did not find theexpected increase in passive tension. Our reported passivetension measurements are somewhat lower than what has beenreported in mice at the same sarcomere length (9), possiblyreflecting the lower N2BA-to-N2B ratio reported in this study.However, it is worth noting that others have found similarN2BA-to-N2B ratios to ours, but those studies did not assesspassive tension (45, 47). These data suggest the presence ofcompensatory mechanisms to offset the isoform induced increasein passive tension. Previous studies have shown that phosphory-lation of titin decreases passive tension (19, 35, 36). Thus it ispossible that in addition to increasing the N2BA-to-N2B ratio,loss of Bmal1 increases basal levels of titin phosphorylation,resulting in a decrease in passive tension even in light of theisoform changes observed. Another potential explanation for the
Fig. 8. Passive tension is not different in Bmal1�/� cardiomyocytes compared with WT controls. Passive tension measurements in single cardiomyocytes fromWT and Bmal1�/� mice at 4 wk (A), 12 wk (B), and 36 wk (C) are shown. Individual data points are binned according to their sarcomere length in bins of 0.05�m to generate graphs (left). Additionally, each curve was fit to an exponential equation: passive tension exp[k*(SL � SL0)], where SL is the sarcomere length,k is the exponent of stiffness, and SL0 is the sarcomere length at zero load. k and SL0 are plotted on the scatter plots (middle and right). Data are means SE;n 20–24 cardiomyocytes from 4 mice/group.
H483DILATED CARDIOMYOPATHY IN Bmal1-DEFICIENT MICE
discrepancy between our titin isoform data and the passive tensiondata might be due to sampling. For our titin isoform determina-tion, we generated a homogenate from a portion of the ventricletaken from the apex of the heart, whereas for the mechanicalmeasurements, we digested the entire heart and performed exper-iments on 5–7 viable cells selected following digestion. Titinisoform expression varies with the region of heart (4). It could bethat by using the apex of the heart, we got an equal representationof all three myocardial layers (epi, mid, and endo), and maybe wehave lost this equal representation of the three layers when usingonly 5–7 cells per mouse for the mechanical studies. Anotherpossibility could be the selective loss of stiffer cells (lowerN2BA-to-N2B ratio) during the cardiomyocyte isolation processfor the mechanical measurements. With the isolation protocol,roughly 30% of cardiomyocytes are lost during the isolation (45a).
In conclusion, this is the first study to show that Bmal1-deficient mice develop a cardiac pathology that starts with atransitory increase in myocardial mass and progresses to dila-tion and failure until their time of death at �36 wk of age. Thesystolic dysfunction observed is associated with early down-regulation of both MHC isoform transcripts, but not withprotein, implicating both transcriptional and post-transcrip-tional changes in myosin gene expression. Bmal1�/� heartsshow loss of the normal sarcomere architecture that is associ-ated with a shift in titin isoform composition toward the stifferisoform. However, titin-based passive tension is not affected,suggesting the presence of other compensatory mechanisms,such as phosphorylation and/or altered intermediate filamentchanges. Collectively, our results suggest that Bmal1 andpotentially the molecular circadian clock play an important rolein maintaining the structural and functional integrity of cardiacmuscle through regulation of titin and MHC.
ACKNOWLEDGMENTS
We thank C. E. Kiper for help with echocardiography; C. Long for helpwith histology; M. G. Engle for help with electron microscopy; M. Mitov forhelp with titin gels; M. Miyazaki for help with myosin gels; and J. J.McCarthy, G. Wolff, and E. A. Schroder for technical advice and support.
GRANTS
This work was supported by NIH Grants R01-AR-055246 andRC1ES018636 (to K. A. Esser) and R01-AR-048816 and R01-HL-098945grants (to J. P. Jin) and by American Heart Association Predoctoral Fellowship10PRE3900047 (to M. Lefta).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
M.L. and K.A.E. conception and design of research; M.L. and H.-Z.F.performed experiments; M.L., K.S.C., H.-Z.F., and J.-P.J. analyzed data; M.L.and K.A.E. interpreted results of experiments; M.L. prepared figures; M.L.drafted manuscript; M.L., K.S.C., J.-P.J., and K.A.E. edited and revisedmanuscript; M.L., K.S.C., H.-Z.F., J.-P.J., and K.A.E. approved final versionof manuscript.
REFERENCES
1. Alvarez JD, Hansen A, Ord T, Bebas P, Chappell PE, GiebultowiczJM, Williams C, Moss S, Sehgal A. The circadian clock protein BMAL1is necessary for fertility and proper testosterone production in mice. J BiolRhythms 23: 26–36, 2008.
2. Andrews JL, Zhang X, McCarthy JJ, McDearmon EL, HornbergerTA, Russell B, Campbell KS, Arbogast S, Reid MB, Walker JR,Hogenesch JB, Takahashi JS, Esser KA. CLOCK and BMAL1 regulate
MyoD and are necessary for maintenance of skeletal muscle phenotypeand function. Proc Natl Acad Sci USA 107: 19090–19095, 2010.
3. Aschoff J. Human circadian rhythms in activity, body temperature andother functions. Life Sci Space Res 5: 159–173, 1967.
4. Bell SP, Nyland L, Tischler MD, McNabb M, Granzier H, LeWinterMM. Alterations in the determinants of diastolic suction during pacingtachycardia. Circ Res 87: 235–240, 2000.
5. Boateng SY, Goldspink PH. Assembly and maintenance of the sarcomerenight and day. Cardiovasc Res 77: 667–675, 2008.
6. Bunger MK, Walisser JA, Sullivan R, Manley PA, Moran SM,Kalscheur VL, Colman RJ, Bradfield CA. Progressive arthropathy inmice with a targeted disruption of the Mop3/Bmal-1 locus. Genesis 41:122–132, 2005.
7. Bunger MK, Wilsbacher LD, Moran SM, Clendenin C, Radcliffe LA,Hogenesch JB, Simon MC, Takahashi JS, Bradfield CA. Mop3 is anessential component of the master circadian pacemaker in mammals. Cell103: 1009–1017, 2000.
8. Campbell KS, Moss RL. SLControl: PC-based data acquisition andanalysis for muscle mechanics. Am J Physiol Heart Circ Physiol 285:H2857–H2864, 2003.
9. Cazorla O, Freiburg A, Helmes M, Centner T, McNabb M, Wu Y,Trombitas K, Labeit S, Granzier H. Differential expression of cardiactitin isoforms and modulation of cellular stiffness. Circ Res 86: 59–67,2000.
miology of idiopathic dilated cardiomyopathy in the elderly: pooled resultsfrom two case-control studies. Am J Epidemiol 143: 881–888, 1996.
12. Curtis AM, Cheng Y, Kapoor S, Reilly D, Price TS, Fitzgerald GA.Circadian variation of blood pressure and the vascular response to asyn-chronous stress. Proc Natl Acad Sci USA 104: 3450–3455, 2007.
13. Durgan DJ, Tsai JY, Grenett MH, Pat BM, Ratcliffe WF, Villegas-Montoya C, Garvey ME, Nagendran J, Dyck JR, Bray MS, GambleKL, Gimble JM, Young ME. Evidence suggesting that the cardiomyo-cyte circadian clock modulates responsiveness of the heart to hypertrophicstimuli in mice. Chronobiol Int 28: 187–203, 2011.
14. Eisenberg BR. Adaptability of ultrastructure in the mammalian muscle.J Exp Biol 115: 55–68, 1985.
15. Feldman MD, Alderman JD, Aroesty JM, Royal HD, Ferguson JJ,Owen RM, Grossman W, McKay RG. Depression of systolic anddiastolic myocardial reserve during atrial pacing tachycardia in patientswith dilated cardiomyopathy. J Clin Invest 82: 1661–1669, 1988.
16. Feng HZ, Biesiadecki BJ, Yu ZB, Hossain MM, Jin JP. RestrictedN-terminal truncation of cardiac troponin T: a novel mechanism forfunctional adaptation to energetic crisis. J Physiol 586: 3537–3550, 2008.
17. Ferreira LF, Moylan JS, Stasko S, Smith JD, Campbell KS, Reid MB.Sphingomyelinase depresses force and calcium sensitivity of the contrac-tile apparatus in mouse diaphragm muscle fibers. J Appl Physiol 112:1538–1545, 2012.
18. Frank D, Kuhn C, Katus HA, Frey N. The sarcomeric Z-disc: a nodalpoint in signalling and disease. J Mol Med 84: 446–468, 2006.
19. Fukuda N, Wu Y, Nair P, Granzier HL. Phosphorylation of titinmodulates passive stiffness of cardiac muscle in a titin isoform-dependentmanner. J Gen Physiol 125: 257–271, 2005.
20. Fuller GA, Bicer S, Hamlin RL, Yamaguchi M, Reiser PJ. Increasedmyosin heavy chain-beta with atrial expression of ventricular light chain-2in canine cardiomyopathy. J Card Fail 13: 680–686, 2007.
21. Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, KingDP, Takahashi JS, Weitz CJ. Role of the CLOCK protein in themammalian circadian mechanism. Science 280: 1564–1569, 1998.
22. Gentry-Smetana S, Redford D, Moore D, Larson DF. Direct effects ofvolatile anesthetics on cardiac function. Perfusion 23: 43–47, 2008.
23. Granzier H, Wu Y, Siegfried L, LeWinter M. Titin: physiologicalfunction and role in cardiomyopathy and failure. Heart Fail Rev 10:211–223, 2005.
27. Ha M, Park J. Shiftwork and metabolic risk factors of cardiovasculardisease. J Occup Health 47: 89–95, 2005.
28. Haddad F, Qin AX, Bodell PW, Zhang LY, Guo H, Giger JM,Baldwin KM. Regulation of antisense RNA expression during cardiacMHC gene switching in response to pressure overload. Am J Physiol HeartCirc Physiol 290: H2351–H2361, 2006.
29. Harding P, Yang XP, Yang J, Shesely E, He Q, LaPointe MC. Geneexpression profiling of dilated cardiomyopathy in older male EP4 knock-out mice. Am J Physiol Heart Circ Physiol 298: H623–H632, 2010.
30. Hasenfuss G, Mulieri LA, Leavitt BJ, Allen PD, Haeberle JR, AlpertNR. Alteration of contractile function and excitation-contraction couplingin dilated cardiomyopathy. Circ Res 70: 1225–1232, 1992.
31. James J, Martin L, Krenz M, Quatman C, Jones F, Klevitsky R,Gulick J, Robbins J. Forced expression of alpha-myosin heavy chain inthe rabbit ventricle results in cardioprotection under cardiomyopathicconditions. Circulation 111: 2339–2346, 2005.
32. Knutsson A. Health disorders of shift workers. Occup Med (Lond) 53:103–108, 2003.
33. Koller M. Health risks related to shift work. An example of time-contingent effects of long-term stress. Int Arch Occup Environ Health 53:59–75, 1983.
34. Kondratov RV, Kondratova AA, Gorbacheva VY, Vykhovanets OV,Antoch MP. Early aging and age-related pathologies in mice deficient inBMAL1, the core component of the circadian clock. Genes Dev 20:1868–1873, 2006.
35. Kruger M, Kotter S, Grutzner A, Lang P, Andresen C, Redfield MM,Butt E, dos Remedios CG, Linke WA. Protein kinase G modulateshuman myocardial passive stiffness by phosphorylation of the titinsprings. Circ Res 104: 87–94, 2009.
36. Kruger M, Linke WA. Protein kinase-A phosphorylates titin in humanheart muscle and reduces myofibrillar passive tension. J Muscle Res CellMotil 27: 435–444, 2006.
37. Laposky A, Easton A, Dugovic C, Walisser J, Bradfield C, Turek F.Deletion of the mammalian circadian clock gene BMAL1/Mop3 altersbaseline sleep architecture and the response to sleep deprivation. Sleep 28:395–409, 2005.
38. Lefta M, Wolff G, Esser KA. Circadian rhythms, the molecular clock,and skeletal muscle. Curr Top Dev Biol 96: 231–271, 2011.
39. McCarthy JJ, Andrews JL, McDearmon EL, Campbell KS, BarberBK, Miller BH, Walker JR, Hogenesch JB, Takahashi JS, Esser KA.Identification of the circadian transcriptome in adult mouse skeletalmuscle. Physiol Genomics 31: 86–95, 2007.
40. McConnell BK, Jones KA, Fatkin D, Arroyo LH, Lee RT, AristizabalO, Turnbull DH, Georgakopoulos D, Kass D, Bond M, Niimura H,Schoen FJ, Conner D, Fischman DA, Seidman CE, Seidman JG.Dilated cardiomyopathy in homozygous myosin-binding protein-C mutantmice. J Clin Invest 104: 1235–1244, 1999.
41. Meyer M, Trost SU, Bluhm WF, Knot HJ, Swanson E, Dillmann WH.Impaired sarcoplasmic reticulum function leads to contractile dysfunctionand cardiac hypertrophy. Am J Physiol Heart Circ Physiol 280: H2046–H2052, 2001.
42. Mosendane T, Raal FJ. Shift work and its effects on the cardiovascularsystem. Cardiovasc J Afr 19: 210–215, 2008.
43. Motulsky H. Fitting Models to Biological Data Using Linear and Non-Linear Regression. A Practical Guide to Curve Fitting. Oxford, UK:Oxford University Press, 2004.
44. Nagueh SF, Shah G, Wu Y, Torre-Amione G, King NM, Lahmers S,Witt CC, Becker K, Labeit S, Granzier HL. Altered titin expression,myocardial stiffness, and left ventricular function in patients with dilatedcardiomyopathy. Circulation 110: 155–162, 2004.
45. Neagoe C, Opitz CA, Makarenko I, Linke WA. Gigantic variety:expression patterns of titin isoforms in striated muscles and consequencesfor myofibrillar passive stiffness. J Muscle Res Cell Motil 24: 175–189,2003.
45a.O’Connell TD, Rodrigo MC, Simpson PC. Isolation and culture of adultmouse cardiac myocytes. Methods Mol Biol 357: 271–296, 2007.
47. Opitz CA, Linke WA. Plasticity of cardiac titin/connectin in heartdevelopment. J Muscle Res Cell Motil 26: 333–342, 2005.
48. Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M,Schultz PG, Kay SA, Takahashi JS, Hogenesch JB. Coordinated tran-scription of key pathways in the mouse by the circadian clock. Cell 109:307–320, 2002.
49. Reiser PJ, Kline WO. Electrophoretic separation and quantitation ofcardiac myosin heavy chain isoforms in eight mammalian species. AmJ Physiol Heart Circ Physiol 274: H1048–H1053, 1998.
50. Roth DM, Swaney JS, Dalton ND, Gilpin EA, Ross J Jr. Impact ofanesthesia on cardiac function during echocardiography in mice. Am J PhysiolHeart Circ Physiol 282: H2134–H2140, 2002.
51. Rudic RD, McNamara P, Curtis AM, Boston RC, Panda S, HogeneschJB, Fitzgerald GA. BMAL1 and CLOCK, two essential components ofthe circadian clock, are involved in glucose homeostasis. PLoS Biol 2:e377, 2004.
52. Schonberger J, Seidman CE. Many roads lead to a broken heart: thegenetics of dilated cardiomyopathy. Am J Hum Genet 69: 249–260, 2001.
53. Seidman JG, Seidman C. The genetic basis for cardiomyopathy: frommutation identification to mechanistic paradigms. Cell 104: 557–567,2001.
54. Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, Wong WH,Weitz CJ. Extensive and divergent circadian gene expression in liver andheart. Nature 417: 78–83, 2002.
55. Sun Y, Yang Z, Niu Z, Wang W, Peng J, Li Q, Ma MY, Zhao Y. Themortality of MOP3 deficient mice with a systemic functional failure.J Biomed Sci 13: 845–851, 2006.
56. Swinnen M, Vanhoutte D, Van Almen GC, Hamdani N, SchellingsMW, D’Hooge J, Van der Velden J, Weaver MS, Sage EH, BornsteinP, Verheyen FK, VandenDriessche T, Chuah MK, Westermann D,Paulus WJ, Van de Werf F, Schroen B, Carmeliet P, Pinto YM,Heymans S. Absence of thrombospondin-2 causes age-related dilatedcardiomyopathy. Circulation 120: 1585–1597, 2009.
57. Talmadge RJ, Roy RR. Electrophoretic separation of rat skeletal musclemyosin heavy-chain isoforms. J Appl Physiol 75: 2337–2340, 1993.
58. Tardiff JC, Hewett TE, Factor SM, Vikstrom KL, Robbins J, Lein-wand LA. Expression of the beta (slow)-isoform of MHC in the adultmouse heart causes dominant-negative functional effects. Am J PhysiolHeart Circ Physiol 278: H412–H419, 2000.
60. Towbin JA, Bowles NE. The failing heart. Nature 415: 227–233, 2002.61. Vitaterna MH, King DP, Chang AM, Kornhauser JM, Lowrey PL,
McDonald JD, Dove WF, Pinto LH, Turek FW, Takahashi JS. Mu-tagenesis and mapping of a mouse gene, Clock, essential for circadianbehavior. Science 264: 719–725, 1994.
62. Warren CM, Krzesinski PR, Campbell KS, Moss RL, Greaser ML.Titin isoform changes in rat myocardium during development. Mech Dev121: 1301–1312, 2004.
63. Warren CM, Krzesinski PR, Greaser ML. Vertical agarose gel electro-phoresis and electroblotting of high-molecular-weight proteins. Electro-phoresis 24: 1695–1702, 2003.
64. Wu J, Bu L, Gong H, Jiang G, Li L, Ma H, Zhou N, Lin L, Chen Z,Ye Y, Niu Y, Sun A, Ge J, Zou Y. Effects of heart rate and anesthetictiming on high-resolution echocardiographic assessment under isofluraneanesthesia in mice. J Ultrasound Med 29: 1771–1778, 2010.
65. Wu Y, Bell SP, Trombitas K, Witt CC, Labeit S, LeWinter MM,Granzier H. Changes in titin isoform expression in pacing-inducedcardiac failure give rise to increased passive muscle stiffness. Circulation106: 1384–1389, 2002.
66. Zakhary DR, Moravec CS, Stewart RW, Bond M. Protein kinase A(PKA)-dependent troponin-I phosphorylation and PKA regulatory sub-units are decreased in human dilated cardiomyopathy. Circulation 99:505–510, 1999.
67. Zhang EE, Kay SA. Clocks not winding down: unravelling circadiannetworks. Nat Rev Mol Cell Biol 11: 764–776, 2010.
68. Zhou YY, Wang SQ, Zhu WZ, Chruscinski A, Kobilka BK, Ziman B,Wang S, Lakatta EG, Cheng H, Xiao RP. Culture and adenoviralinfection of adult mouse cardiac myocytes: methods for cellular geneticphysiology. Am J Physiol Heart Circ Physiol 279: H429–H436, 2000.
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