Exercise training during diabetes attenuates cardiac ryanodinereceptor dysregulation
Chun-Hong Shao,1 Xander H. T. Wehrens,4 Todd A. Wyatt,2 Sheeva Parbhu,1 George J. Rozanski,3
Kaushik P. Patel,3 and Keshore R. Bidasee1,4
Departments of 1Pharmacology and Experimental Neuroscience, 2Environmental, Agricultural, and Occupational Health,and 3Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska; and 4Departmentof Molecular Physiology & Biophysics and Medicine, Baylor College of Medicine, Houston, Texas
Submitted 24 September 2008; accepted in final form 7 January 2009
Shao CH, Wehrens XH, Wyatt TA, Parbhu S, Rozanski GJ, PatelKP, Bidasee KR. Exercise training during diabetes attenuates cardiac ryano-dine receptor dysregulation. J Appl Physiol 106: 1280–1292, 2009. Firstpublished January 8, 2009; doi:10.1152/japplphysiol.91280.2008.—Thepresent study was undertaken to assess the effects of exercise training(ExT) initiated after the onset of diabetes on cardiac ryanodinereceptor expression and function. Type 1 diabetes was induced inmale Sprague-Dawley rats using streptozotocin (STZ). Three weeksafter STZ injection, diabetic rats were divided into two groups. Onegroup underwent ExT for 4 wk while the other group remainedsedentary. After 7 wk of sedentary diabetes, cardiac fractional short-ening, rate of rise of left ventricular pressure, and myocyte contractilevelocity were reduced by 14, 36, 44%, respectively. SpontaneousCa2� spark frequency increased threefold, and evoked Ca2� releasewas dyssynchronous with diastolic Ca2� releases. Steady-state type 2ryanodine receptor (RyR2) protein did not change, but its response toCa2� was altered. RyR2 also exhibited 1.8- and 1.5-fold increases inphosphorylation at Ser2808 and Ser2814. PKA activity was reduced by75%, but CaMKII activity was increased by 50%. Four weeks of ExTinitiated 3 wk after the onset of diabetes blunted decreases in cardiacfractional shortening and rate of left ventricular pressure development,increased the responsiveness of the myocardium to isoproterenolstimulation, attenuated the increase in Ca2� spark frequency, andminimized dyssynchronous and diastolic Ca2� releases. ExT alsonormalized the responsiveness of RyR2 to Ca2� activation, attenuatedincreases in RyR2 phosphorylation at Ser2808 and Ser2814, and nor-malized CaMKII and PKA activities. These data are the first to showthat ExT during diabetes normalizes RyR2 function and Ca2� releasefrom the sarcoplasmic reticulum, providing insights into mechanismsby which ExT during diabetes improves cardiac function.
type 1 diabetes; calcium sparks; calcium transients; phosphorylation;hemodynamics
WE ARE IN THE MIDST of an epidemic of diabetes mellitus. Morethan 240 million individuals are afflicted worldwide, andexperts predict this number will increase to nearly 380 millionby the year 2025 (28). In the United States, about 18 millionindividuals (7.8% of the population) have been diagnosed withdiabetes, and another 6 million have the syndrome but areunaware that they have it (1). Diabetes mellitus is an estab-lished risk factor for adverse cardiovascular events, includingheart failure, which occurs at rates three to five times higher inthis group than in the general population (21). To date, theetiology underlying diabetes-induced reductions in myocyteand cardiac contractility remains incompletely understood.
However, numerous studies including work from our labora-tory suggest that these defects stem in part from perturbation inintracellular Ca2� cycling (3, 11, 39, 44).
The release of Ca2� from the internal sarcoplasmic reticu-lum via type 2 ryanodine receptor (RyR2) is an integral step inthe cascade of events leading to cardiac muscle contraction (4).Our group (44) and others (54) recently showed that ventricularmyocytes isolated from streptozotocin (STZ)-induced type 1diabetic rat hearts exhibited increased frequency of spontane-ous Ca2� sparks. Our group (44) also showed that evokedrelease of Ca2� from the sarcoplasmic reticulum (SR) wasdyssynchronous with increased diastolic Ca2� release. Fromthese and other studies, we hypothesized that alteration in thesensitivity of RyR2 to Ca2� activation (i.e., RyR2 dysregula-tion) during chronic diabetes is responsible in part for slowingin rates of myocyte and cardiac contractility (6, 7, 8, 39, 44,54). In longer term or more severe experimental diabetes,reduction in steady-state levels of RyR2 (and other Ca2�
cycling proteins) also contributes (8, 11, 26, 38). To date,molecular mechanisms underlying RyR2 dysregulation duringchronic diabetes remain incompletely understood. However,alterations in the sensitivity of RyR2 to Ca2� activation couldresult from increases in phosphorylation at PKA (Ser2808) andCaMKII (Ser2808 and Ser2814) sites (25, 49, 50). Oxidation ofRyR2 by reactive oxygen species (ROS) and/or reactive car-bonyl species also may contribute (9, 20, 53). Functionaluncoupling of RyR2 from L-type Ca2� channels on the invag-inated T-tubule membranes also could be responsible in partfor the dyssynchronous Ca2� release (46).
Clinical as well as experimental studies have consistentlydemonstrated that exercise training (ExT) is one of the mosteffective strategies for slowing the development of cardiomy-opathy and for reducing the incidence of cardiovascular mor-bidity and mortality during diabetes (40, 41, 43). One of themost significant benefits of ExT is its ability to maintaincardiac output by blunting diabetes-induced bradycardia andthe reduction in force of myocardial contractility. These im-provements have been attributed to normalization of sympa-thetic outflow (neurohormonal), an increase in the responsive-ness of the myocardium to autonomic stimulation (13, 43), andan increase in expression and function of �-adrenergic receptorand/or signaling (5, 14). ExT also was shown to normalizecirculating levels of catecholamines, angiotensin II, vasopres-sin, neuropeptide Y, atrial natriuretic peptides, and proinflam-
Address for reprint requests and other correspondence: K. R. Bidasee,985800 Nebraska Medical Center, Durham Research Center, DRC 3047,Omaha, NE 68198-5800 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
J Appl Physiol 106: 1280–1292, 2009.First published January 8, 2009; doi:10.1152/japplphysiol.91280.2008.
matory mediators such as soluble intracellular adhesion mole-cule, vascular cell adhesion molecule, monocyte chemotacticadhesion molecule-1, and tumor necrosis factor-� (2, 12, 15,16, 30, 33, 36). Although a few studies have shown that ExTnormalizes expression of Ca2� cycling proteins during heartfailure (18, 19, 31, 32, 42), to date the effect of ExT duringdiabetes on myocyte intracellular Ca2� cycling and the func-tion of SR proteins remain poorly characterized. Earlier, Witc-zak et al. (51) found that ExT prevented Ca2� dysregulation incoronary smooth muscle from diabetic dyslipidemic Yucatanswine and attributed this in part to a reduction in diabetes-induced increase in sarco(endo)plasmic reticulum Ca2�-ATPase(SERCA2b) expression. More recently, Howarth et al. (27)found that time to peak myocyte Ca2� transient was prolongedby light and moderate exercise initiated 2 mo after the onset ofdiabetes.
The present study was undertaken to assess whether ExTinitiated 3 wk after the onset of experimental type 1 diabetes isable to blunt increases in spontaneous and diastolic Ca2�
releases, prevent dyssynchronous Ca2� release, and normalizethe sensitivity of RyR2 to Ca2� activation. We also investi-gated whether ExT is able to attenuate increases in phosphor-ylation of RyR2 at Ser2809 and Ser2814.
MATERIAL AND METHODS
Chemicals and Drugs
Mouse monoclonal RyR2, SERCA2, and phospholamban (PLN)antibodies were obtained from Affinity Bioreagents (Golden, CO).Phospho-CaMKII (Thr286/287, clone 22B1) was obtained from Cay-man Chemicals (Ann Arbor, MI). Actin antibodies (C-11) and anti-CaMKII� (A-17) were obtained from Santa Cruz Biotechnology(Santa Cruz, CA). The phospho-RyR2(Ser2808) (1:1,000) and phos-pho-RyR2(Ser2814) (1:1,000) phosphoepitope-specific antibodieswere custom generated using peptide C-RTRRI-(pS)-QTSQV, corre-sponding to the PKA phosphorylation site region at Ser2808 on RyR2,and peptide CSQTSQV-(pS)-VD, corresponding to CaMKII phos-phorylation of RyR2 at Ser2814. Type 2 collagenase was obtained fromWorthington Biomedical (Lakewood, NJ). Dulbecco’s modified Ea-gle’s medium (DMEM) and F-12 supplement were obtained fromInvitrogen (Carlsbad, CA). Heptapeptide substrate (LRRASLG) forthe PKA activity assay was obtained from Bachem Biosciences(Torrance, CA). P-81 phosphocellulose papers were obtained fromWhatman (Hillsboro, OR). Other reagents and solvents used were ofanalytical grade.
Induction and Verification of Experimental STZ-Induced Diabetes
Animal procedures used for the study adhered to the AmericanPhysiological Society’s “Guiding Principles in the Care and Use ofAnimals” and were in accordance with the Institute for LaboratoryResearch Guide for the Care and Use of Laboratory Animals (37).Animal protocols also were approved by the University of NebraskaMedical Center Institutional Animal Care and Use Committee. Thirty-six male Sprague-Dawley rats (200–210 g) were purchased fromSasco Breeding Laboratories (Omaha, NE). Animals were housedwith a 12:12-h light-dark cycle at ambient 22°C, 30–40% relativehumidity, and were given laboratory chow and tap water ad libitum.After acclimatization for 1 wk, rats were assigned randomly to one oftwo groups: control or STZ-diabetic. STZ-diabetic rats received asingle injection of STZ (55 mg/kg ip; Sigma-Aldrich, St. Louis, MO)in a 2% solution of cold 0.1 M citrate buffer (pH 4.5). Control ratswere injected with a similar volume of citrate buffer only. Blood sugarlevels of animals were maintained between 19.2 and 25 mmol (350–450 mg/dl) throughout the study.
Exercise Training Protocol
Starting 1 wk after injection of STZ (or vehicle), rats were removedfrom their cages and placed on a slow-moving (2 m/min) treadmill for10–15 min/day to acclimate them to the movement of the treadmill.This acclimatization continued for two more weeks. Twenty-one daysafter injection of STZ (or vehicle), control and diabetic animals wererandomly subdivided into two groups each. One group of control andone group of diabetic rats continued on the treadmill for 10–15min/day, 5 days/wk, at a speed of 2 m/min for the duration of theprotocol. These rats were designated sedentary (control or diabetic).The other groups of control and diabetic animals were subjected toExT. During week 1, rats were placed on a treadmill for 15–20min/day at a speed of 15–20 m/min and a grade of 0%. During week2, the speed and duration of ExT were gradually increased to amaximum rate of 25 m/min and a duration of 60 min/day. Duringweek 3, the intensity of the ExT was gradually increased byincreasing the grade of the treadmill running platform to 5% whilemaintaining the speed and duration. During week 4 of ExT (week7 of protocol), the speed was maintained at 25 m/min, the grade at5%, and the duration at 60 min.
Assessment of Cardiac Function
M-mode echocardiography. Sixteen hours after the last bout ofExT, M-mode echocardiography was performed on all rats within thefour groups (sedentary control, sedentary STZ-diabetic, ExT control,and ExT STZ-diabetic). For this, rats were lightly anesthetized withketamine-acepromazine (91 mg/kg ip ketamine and 1.8 mg/kg ipacepromazine), and an Acuson Sequoia 512C ultrasound system(Siemens) with an Acuson 15L8 probe was used to measure heart rate,left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic volume(LVEDV), and left ventricular end-systolic volume (LVESV). Percentfractional shortening (FS) was calculated using the equation FS �[(LVEDD � LVESD)/LVEDD] � 100. Percent ejection fraction (EF)was calculated using the equation EF � [(LVEDV � LVESV)/LVEDV] � 100.
In vivo hemodynamics. Heart rate, peak left ventricular pressure(LVP), left ventricular end-diastolic pressure (LVEDP), and rates ofchange of left ventricular pressures (�dP/dt) were also evaluated toascertain the effect of ExT on cardiac hemodynamics. For this, ratswere anesthetized with Inactin (20 mg/kg ip), and a Millar catheter(Millar Instruments, Houston TX) containing a pressure transducerwas introduced into the left ventricle via the right carotid artery aspreviously described (5). Another catheter was inserted via the rightfemoral vein for administration of isoproterenol. Basal cardiac hemo-dynamic parameters were then measured for the four groups of rats.After basal parameters were assessed, a bolus dose of 0.1 g/kgisoproterenol was administered into the right femoral vein to assessthe responsiveness of the heart to �-adrenoceptor stimulation. APowerLab data acquisition system (ADInstuments, Colorado Springs,CO) was used for acquiring data. Microsoft Excel (Seattle, WA) andGraphPad Prism 4.0 (San Diego, CA) were used for analysis of data.
Sample Collection
Thirty minutes after isoproterenol injection (for in vivo hemody-namic measurements), animals were injected with a single lethal doseof Inactin (150 mg/kg ip). Abdominal cavities were opened, and bloodsamples were collected via left renal arteries. Chest cavities were thenopened, and hearts were removed and quick-frozen, either by beingdropped into liquid nitrogen or embedded in crushed dried ice, orplaced in Krebs-Henseleit buffer for isolation of ventricular myocytes.Soleus muscles from hind legs were also excised, quick-frozen, andstored at �80°C until use.
1281EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
Citrate synthase activity in soleus muscle was measured spectro-photometrically, employing the method described by Srere (47). Allmeasurements were performed in duplicate under the same experi-mental setting at 20–22°C. Citrate synthase activities were normal-ized to total protein content and are reported as micromoles permilligram of protein per minute.
Isolation of Myocytes
Ventricular myocytes were isolated as described previously (44).Briefly, 10 min before injection of Inactin (150 mg/kg ip), rats wereinjected with heparin (1,000 U/kg ip). Hearts were removed andperfused with collagenase, and left ventricular myocytes were iso-lated. Cells were used within 5–6 h after isolation.
Myocyte Contractile Kinetics
Cells were placed in a chamber mounted on the stage of an invertedmicroscope (Zeiss X-40; Gottingen, Germany) at room temperature(22–24°C) and field stimulated (10 V) at a frequency of 0.5 Hz for 10ms in duration, using a pair of platinum wires. Contractile kinetics ofmyocytes was measured using a high-speed video edge-detectionsystem (IonOptix, Milton, MA) (44).
Measurement of Spontaneous Ca2� Release
Spontaneous Ca2� releases (Ca2� sparks) were assessed as de-scribed previously (44). Briefly, ventricular myocytes in DMEM-F12were incubated for 1 h at 37°C in petri dishes containing glasscoverslips that had been previously coated with laminin. After incu-bation, unbound cells were gently removed by suction, and Tyrodesolution (140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 1.0 mMCaCl2, 10 mM HEPES, 0.25 mM NaH2PO4, and 5.6 mM glucose, pH7.3) was added. Cells were then loaded with fluo-3 (5 M) for 30 minat 37°C. Spontaneous Ca2� sparks were recorded in line-scan modewith the use of a Zeiss LSM 410 confocal microscope equipped withan argon-krypton laser (25 mW, 5% intensity) with a �60 objective.Spark characteristics were analyzed using LSM 5 Meta (Zeiss),GraphPad Prism 4.0, and Microsoft Excel.
Measurement of Evoked Ca2� Release
Evoked Ca2� release was assessed as described previously (44).Briefly, cells bound to laminin-coated coverslips in DMEM-F12 wereloaded with fluo-3 (5 M) for 30 min at 37°C. At the end of theincubation, cells were washed to remove extracellular fluo-3 andplaced in a chamber on the stage of the confocal microscope. Cellswere then field stimulated at 0.5 Hz (10 V for 10 ms), and changes influorescence intensities (F) were determined. Fluo-3 was excited bylight at 488 nm, and fluorescence was measured at wavelengths of�515 nm. LSM 5 Meta, Prism 4.0, and Microsoft Excel were used foranalyzing rates of Ca2� rise (linear regression) and decay constants(one-phase exponential decay).
Measurement of SR Ca2� Load
Myocytes were loaded with fura-2 AM in DMEM-F12 mediumcontaining 1.2 mM Ca2�. After loading, cells were washed to removeextracellular fura-2 AM and then pulse stimulated four times at afrequency of 0.025 Hz. Forty seconds after the last stimulation, cellswere challenged with 10 mM caffeine, and the rate and amplitude ofCa2� release were recorded using a dual-excitation fluorescencephotomultiplier system (Image Master Fluorescence Microscope;Photo Technology International USA).
Assessing Ca2� Sensitivity of RyR2
The responsiveness of RyR2 to Ca2� activation and deactivationwas assessed using [3H]ryanodine binding assays (6–9, 44). For this,
SR membrane vesicles (0.1 mg/ml) from sedentary and ExT animalswere incubated in binding buffer (500 mM KCl, 20 mM Tris �HCl, 5mM reduced glutathione, and 100 M EGTA, pH 7.4) for 2 h at 37°Cwith 6.7 nM [3H]ryanodine and increasing amounts of Ca2� (0.1 nMto 5 mM). After incubation, vesicles were filtered and washed, and theamount of [3H]ryanodine bound to RyR2 was determined using liquidscintillation counting. Nonspecific binding was determined simulta-neously by incubating vesicles with 1,000 nM unlabeled ryanodine.
Determination of Total and Phosphorylated RyR2(Ser2809 and Ser2814)
Membrane vesicles were prepared from sedentary and ExT rathearts using the procedure described previously (6–9, 44) with theinclusion of phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich)in the isolation buffer. Western blot analyses were then used todetermine relative levels of total and phosphorylated RyR2 (Ser2808
and Ser2814) in samples. Primary RyR2 antibodies were used at1:2,500 and 1:1,000 dilutions for phosphorylated RyR2. Membraneswere electrophoresed using 4–15% linear gradient polyacrylamidegels at 150 V for 2.5 h.
Determination of SERCA2 and PLN
Because SERCA2 is intimately involved in regulating the SR Ca2�
load, Western blot analyzes also were performed to determine steady-state levels of SERCA2 and its intrinsic regulator, PLN. For SERCA2assessment, membranes were electrophoresed using 4–15% lineargradient polyacrylamide gels at 150 V for 2.5 h with 1:2,000 dilutionof the primary antibody. For PLN, membranes were electrophoresedusing 15% linear gradient polyacrylamide gels at 150 V for 1.5 h with1:2,000 dilution of the primary antibody.
PKA Activity
Intrinsic PKA activity was determined using a modification ofprocedures previously described (52). Briefly, left ventricular homog-enates (20 l) were added to 50 l of reaction mix {130 M PKAsubstrate heptapeptide (LRRASLG), 0.9 mg/ml BSA, 0.2 mM IBMX,20 mM Mg-acetate, and 0.2 mM �-[32P]ATP in a 40 mM Tris �HClbuffer, pH 7.5} and incubated at 30°C for 10 min. Experiments alsowere performed in the presence of 10 M cAMP to assess maximumactivatable PKA activity in each sample. Incubations were halted byspotting 60 l of each sample onto P-81 phosphocellulose papers.Papers were then washed five times for 5 min each in 75 mMphosphoric acid, washed once in ethanol, dried, and analyzed byscintillation spectroscopy. Kinase activity is expressed as picomolesof phosphate incorporated per minute per milligram of protein. Ex-periments were conducted three separate times (n � 3), and the resultsare expressed as means � SE for each data point.
CaMKII Activity
The extent of phosphorylation at Thr287 on CaMKII was measuredand used as an indirect measure of total cytoplasmic CaMKII activity.For this, Western blot analyses were conducted to determine totalCaMKII protein as well as phospho-CaMKII(Thr287). Phospho-CaMKII(Thr287) antibodies were used at 1:1,000 dilution.
Statistical Analysis
Differences among values from each of the four groups (control,STZ-induced, ExT control, and ExT diabetic) were evaluated usingtwo-way ANOVA, employing Prism 4 (GraphPad Software, SanDiego, CA). Data are means � SE. Results were considered signifi-cantly different if P 0.05.
1282 EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
The general characteristics of the animals used in this studyare shown in Table 1. Diabetic animals fed normally, but theirbody weights were significantly less (P 0.05) than that ofcontrols. Sedentary animals tended to have higher body massesthan their ExT counterparts, but mean difference was not largeenough to attain statistical significance. Throughout the proto-col, mean blood glucose levels of sedentary and ExT diabeticanimals were �22.0 � 2.5 mM. Glycosylated hemoglobinlevels of sedentary and ExT diabetic animals were �8%.Plasma insulin levels were lower in sedentary and ExT diabeticanimals (P 0.05). Hearts from sedentary and ExT diabeticanimals were significantly smaller than those from sedentarycontrols (P 0.05), and this impacted on heart-to-body weightratios (Table 1). ExT increased citrate synthase activity by�75% (Table 1) in both control and diabetic animals.
In Vivo Left Ventricular Function
M-mode echocardiography. Compared with nondiabeticcontrols, STZ-diabetic rats were bradycardic (416.5 � 17.6 vs.304.8 � 13.1 beats/min; Fig. 1). Sedentary diabetic animalshad significant reductions in EF and FS compared with seden-tary nondiabetic controls. QRS intervals also were significantlygreater in sedentary diabetic animals (P 0.05). MeanLVEDD and LVESD also were significantly larger in seden-tary diabetic animals than in sedentary control animals. Dia-betes did not alter mean aortic diameter. ExT attenuated thebradycardia and blunted increases in LVEDD, LVESD, andQRS and QT intervals. ExT also blunted the decrease in percentFS. There was no significant difference in mean EF betweensedentary and ExT diabetic animals. The ExT protocol used in thisstudy did not alter cardiac parameters in control animals.
In vivo hemodynamics. Consistent with echocardiographicdata, mean basal heart rate of Inactin-anesthetized sedentarydiabetic animals was significantly lower than that of sedentarycontrol animals (296 � 14.5 vs. 348 � 9.3 beats/min, respec-tively; P 0.05). Mean peak LVP and �dP/dt also weresignificantly lower in sedentary STZ-diabetic animals than insedentary control animals (Fig. 2, A and B). Diabetes alsoincreased mean LVEDP (8.1 � 1.9 vs. 1.2 � 0.9 mmHg, P 0.05). ExT did not significantly change basal peak LVP incontrol animals, but it significantly blunted the reduction in peakLVP induced by diabetes and attenuated the increase inLVEDP to 4.2 � 0.4 mmHg (P 0.05). Although ExT did notattenuate the reduction in basal �dP/dt, it significantly in-creased �dP/dt (Fig. 2B, bottom left).
ExT during diabetes also significantly enhanced the respon-siveness of hearts to isoproterenol stimulation (Fig. 2B). Wheninjected with 0.1 g/kg isoproterenol, mean peak LVP inhearts of sedentary control animals increased to 139.9 � 5.0mmHg, whereas that of sedentary STZ-diabetic animals was124.6 � 8.0 mmHg. Values for �dP/dt following isoproterenolstimulation also increased (Fig. 2B). Similar trends also wereobserved following injection with 0.5 g/kg isoproterenol(data not shown). In these studies, ExT did not significantlyalter �dP/dt in control animals (Fig. 2B).
Myocyte Contractile Kinetics
As reported previously (44), our laboratory found thatduring isolation, myocytes from sedentary diabetic rats wereless tolerant to Ca2� reconstitution compared with myocytesfrom sedentary control rats. ExT minimized this Ca2� in-tolerance and increased the yield of myocytes to near that ofcontrol animals (�65%). There were no significant differ-ences in the mean lengths of myocytes isolated from sed-entary control, sedentary diabetic, ExT control, or ExTdiabetic rat hearts (Fig. 3). We did not measure cell volume.When cells were stimulated at a frequency of 0.5 Hz,velocities of cell shortening and relengthening were signif-icantly slower in diabetic myocytes than in sedentary con-trol myocytes. Extent of shortening also was 26% less inmyocytes isolated from sedentary STZ-diabetic animals,and times to 50% peak myocyte shortening and relengthen-ing were longer. ExT during diabetes blunted the reductionin contractile kinetics induced by diabetes. Velocities ofcontraction and relaxation and extent of cell shortening inmyocytes from ExT control animals were not significantlydifferent from those of sedentary control animals.
Spontaneous Ca2� Release From SR
Figure 4 shows representative line-scan images of spontaneousCa2� release in myocytes from sedentary control (A), sedentarySTZ-diabetic (B), ExT control (C), and ExT STZ-diabetic animals(D). Greater than 80% of myocytes from sedentary control rathearts exhibited spontaneous Ca2� releases, whereas �50% ofmyocytes from sedentary diabetic rats showed spontaneous Ca2�
sparks. Interestingly, those diabetic myocytes that did generatespontaneous Ca2� releases did so at a frequency that was three-fold higher than that of sedentary control myocytes (Fig. 4B). Theduration and full width at half maximum of Ca2� sparks insedentary diabetic myocytes were similar to those in sedentarycontrols, but rate of Ca2� rise was slower and peak Ca2� ampli-tude was less. Greater than 75% of myocytes from ExT STZ-
Table 1. General characteristics of animals used in this study
Parameters Sedentary Control Sedentary STZ-Diabetic Exercise-Trained Control Exercise-Trained STZ-Diabetic
Values are means � SE of parameters measured in sedentary control, sedentary streptozotocin (STZ)-diabetic, exercise-trained control, and exercise-trainedSTZ-diabetic rats (n � 9 per group). *P 0.05, significantly different from sedentary control. †P 0.05, significantly different from sedentary STZ-diabetic.
1283EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
diabetic animals generated spontaneous Ca2� sparks, albeit at alower frequency. ExT did not change the frequency of spontane-ous Ca2� spark generation in control animals, but the sparks weresomewhat brighter (increase Ca2� amplitude).
Evoked Ca2� Release
Representative evoked intracellular Ca2� transients areshown in Fig. 5. When cells were stimulated at a frequency of0.5 Hz, the rate of evoked Ca2� release, peak Ca2� transient
amplitude (F), and Ca2� decay times were significantly (P 0.05) slower in sedentary diabetic rat myocytes than in seden-tary control myocytes. Myocytes from sedentary diabetic ani-mals also exhibited diastolic Ca2� release in between pulses(green arrows in Fig. 5B). About 50% of ventricular myocytesfrom sedentary diabetic rat hearts, primarily those that exhib-ited increased Ca2� spark frequency, also showed dyssynchro-nous (nonuniform) Ca2� releases (white arrow in Fig. 5B). ExTblunted diabetes-induced slowing in rates of Ca2� rise and
Fig. 1. Representative echocardiograms (top) and hemodynamic parameters (bottom) from sedentary control (C), sedentary streptozotocin (STZ)-diabetic (D),exercise-trained control (ExT-C), and ExT STZ-diabetic (ExT-D) rats. Animals were lightly anesthetized with 0.5 ml of a cocktail containing ketamine (100mg/ml) and acepromazine (10 mg/ml). Three loops of M-mode echocardiography were captured for each animal. Values are means � SE (n � 9). LVEDD, leftventricular end-diastolic diameter. LVESD, left ventricular end-systolic diameter. *P 0.05, significantly different from sedentary and ExT controls. **P 0.05, significantly different from sedentary STZ-diabetic.
Fig. 2. A: peak left ventricular pressure inhearts from sedentary control, sedentary STZ-diabetic, ExT control, and ExT STZ-diabeticrats. Animals were lightly anesthetized withInactin (20 mg/kg ip), and an F-2 microma-nometer-tipped catheter (Millar Instruments,Houston, TX) was inserted via the right carotidartery into the left ventricle. Values aremeans � SE (n � 9). B: rates of change of leftventricular pressure (�dP/dt) with and withoutisoproterenol (ISO) stimulation. Values aremeans � SE (n � 9). *P 0.05, significantlydifferent from sedentary control. **P 0.05,significantly different from sedentary STZ-di-abetic.
1284 EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
decay and reduction in Ca2� transient amplitude. Myocytesfrom ExT STZ-diabetic animals did not exhibit diastolic Ca2�
release in between pulses (compare Fig. 5, B and D). ExT didnot significantly alter the kinetics of Ca2� transients in nondi-abetic animals.
SR Ca2� Load
As shown in Fig. 6, basal intracellular Ca2� levels ([Ca2�]i)in sedentary diabetic rat myocytes were higher than in seden-tary control myocytes (340 nm/380 nm fluorescence ratio �0.13 � 0.01 vs. 0.07 � 0.01, basal [Ca2�]i in sedentarydiabetic myocytes � 119.1 � 1.3 nM, and basal [Ca2�]i insedentary control myocytes � 83.1 � 1.2 nM, n � 6). Theamplitudes of depolarization-evoked Ca2� transients in seden-tary diabetic myocytes also were significantly smaller than insedentary control myocytes (compare responses at small ar-rows in Fig. 6, A and B). When challenged with 10 mMcaffeine, sedentary diabetic myocytes released 15.4 � 3.1%less Ca2� from the SR compared with sedentary control myo-cytes (P 0.05). Rate of Ca2� rise and time to 50% Ca2�
decay (T50 decay) also were significantly slower in sedentarydiabetic myocytes than in sedentary control myocytes (rate ofCa2� rise � 0.014 � 0.001 vs. 0.040 � 0.002 fluorescenceratio/s and T50 decay � 17.31 � 1.2 vs. 8.66 � 0.97 s, P
0.05). ExT blunted the rise in basal [Ca2�]i, increased theamplitudes of evoked Ca2� releases, increased the rate andamplitude of caffeine-releasable Ca2�, and increased T50 decay(Fig. 6D). ExT did not significantly alter the amplitude ofevoked and caffeine-releasable Ca2� from control myocytes(Fig. 6C).
Ca2� Sensitivity of RyR2
After normalization to �-actin, no significant differenceswere observed in steady-state levels of RyR2 protein in heartsfrom sedentary control, sedentary STZ-diabetic, ExT control,and ExT STZ-diabetic rats (Fig. 7, B and C, middle autoradio-grams). However, RyR2 from sedentary diabetic animals ex-hibited altered responsiveness to Ca2� activation and boundless [3H]ryanodine at peak [Ca2�] (200–300 M, Fig. 7A).Half-maximal Ca2� activation of diabetic RyR2 also occurredat lower [Ca2�] for sedentary diabetic (EC50 activation �23.3 � 5.2 M for sedentary STZ-diabetic vs. 72.4 � 8.1 Mfor sedentary control). In this study we also found that RyR2from sedentary diabetic animals was less sensitive to Ca2�
deactivation (EC50 deactivation � 3,102.1 � 290.1 M Ca2�
for sedentary diabetic vs. 2,091.1 � 350.5 M Ca2� forsedentary control). ExT normalized the sensitivity of RyR2 toCa2� activation and deactivation. ExT did not significantly
Fig. 3. Representative evoked contractile kinetics (A) ofventricular myocytes isolated from sedentary control, sed-entary STZ-diabetic, ExT control, and ExT STZ-diabetic rathearts. Values shown in B are means � SE (n � 116 cells).*P 0.05, significantly different from sedentary and ExTcontrols. **P 0.05, significantly different from sedentarySTZ-diabetic.
1285EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
Fig. 4. Representative line-scan images showing spontaneous Ca2� releases in isolated myocytes from sedentary control (A), sedentary STZ-diabetic (B), ExTcontrol (C), and ExT STZ-diabetic rat hearts(D). Graphs below each panel show the fluorescence profile of the spark highlighted above with the green arrow.A spatiotemporal profile of Ca2� sparks is shown in the chart at bottom. Values are mean � SE (n � 43 cells). (F � Fo)/Fo, change in fluorescence intensityrepresenting peak Ca2� transient amplitude; T50 decay, time to 50% Ca2� decay.*P 0.05, significantly different from sedentary and ExT control. **P 0.05,significantly different sedentary STZ-diabetic.
1286 EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
alter the responsiveness of RyR2 from control nondiabeticanimals to Ca2� activation or deactivation.
Relative Levels of RyR2 Phosphorylation at Ser2808
and Ser2814
One likely mechanism for increased sensitivity of RyR2 toCa2� activation is an increase in its extent of phosphorylation (25,34, 49). In the present study we investigated phosphorylation atSer2808 and Ser2814. Consistent with prior reports (44, 54), wefound increased phosphorylation of RyR2 at Ser2808 (75.2 � 1.5%over control) in hearts of sedentary STZ-diabetic rats (Fig. 7B). Inthis study we also found increased phosphorylation of RyR2 atSer2814 (50.1 � 8.1 over control, Fig. 7C). Increases in phosphor-ylation of RyR2 at Ser2808 and Ser2814 were attenuated with ExT.
RyR2 from ExT control animals also consistently showed ele-vated levels of phosphorylation at Ser2808 and Ser2814 (Fig. 7, Band C). We also measured and found reduced levels of calstabin2on RyR2 from sedentary STZ-diabetic animals, and ExT blunteddissociation of calstabin2 from RyR2 (Fig. 7B, bottom autoradio-gram). Interestingly, the steady-state level of RyR2 protein wasnot significantly different between sample types.
Relative Levels of SERCA2 and PLN
As shown above, reuptake of Ca2� into SR followingelectrical and caffeine stimulations was slower in myocytesfrom diabetic rat hearts than in myocytes from control animals.These data prompted us to investigate steady-state levels ofSERCA2 and its regulator protein, PLN. As shown in Fig. 7D,
Fig. 5. Representative line-scan images showing electrically evoked global Ca2� transients in isolated myocytes from sedentary control (A), sedentarySTZ-diabetic (B), ExT control (C), and ExT STZ-diabetic rat hearts (D). White arrow in B shows dyssynchronous evoked Ca2� release; green arrows showdiastolic Ca2� releases. Chart at bottom shows Ca2� transient characteristics. Values are means � SE (n � 53 cells) *P 0.05, significantly different fromsedentary and ExT control. **P 0.05, significantly different from sedentary STZ-diabetic.
1287EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
although there was a trend toward a reduction in steady-statelevels of SERCA2 in sedentary diabetic rat hearts (usinglow-dose STZ), the difference was not large enough to attainstatistical significance (86.2 � 12.3 vs. 100.0 � 4.0, n � 7
animals). We did not detect any significant change in eithermonomeric (unphosphorylated) or pentameric (phosphory-lated) PLN levels in hearts of control and STZ-diabetic ani-mals. Interestingly, hearts from ExT control animals consis-
Fig. 6. Representative caffeine-induced Ca2�
transients in ventricular myocytes isolated fromsedentary control (A), sedentary STZ-diabetic(B), ExT control (C), and ExT STZ-diabetic rathearts (D). Small arrows indicate electricallyevoked stimulation; larger arrows indicate ap-plication of caffeine. These experiments weredone 6 times, and average data are given in thetext.
Fig. 7. A: Ca2�-sensitive binding of [3H]ry-anodine to type 2 ryanodine receptor (RyR2)from sedentary control, sedentary STZ-dia-betic, ExT control, and ExT STZ-diabetic rathearts. Data are means � SE from at least 5different sarcoplasmic reticulum (SR) mem-brane preparations. B: representative West-ern blots of total RyR2 (tRyR2), phospho-RyR2 (pRyR2) (Ser2808), and calstabin2 lev-els. Graph at bottom shows mean � SE ofrelative pRyR2 (Ser2808) levels obtainedfrom 5 separate preparations. C: representa-tive Western blots of tRyR2, pRyR2(Ser2814), and �-actin (internal reference)levels. Graph at bottom shows means � SEof relative pRyR2 (Ser2814) levels obtainedfrom 5 separate preparations. D: representa-tive Western blots of sarco(endo)plasmic re-ticulum Ca2�-ATPase (SERCA2) levels (topblots) obtained using 2 different amounts ofmembrane vesicular proteins for loading gels(5 and 20 g). Graph at bottom showsmeans � SE of relative SERCA2 levelsobtained from 7 separate sets of prepara-tions. Bottom blots show pentameric andmonomeric phospholamban (PLN) levels.*P 0.05, significantly different from sed-entary control. **P 0.05, significantly dif-ferent from sedentary STZ-diabetic.
1288 EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
tently expressed significantly higher levels of SERCA2 (Fig.7D, graph), and pentameric PLN levels (128.2 � 6.4% oversedentary controls) also were higher. Monomeric PLN levels,however, remained unchanged. ExT during diabetes did notalter steady-state levels of SERCA2 and PLN (monomeric orpentameric).
PKA and CaMKII Activities
Maximum activatable as well as intrinsic PKA activitieswere assessed in the presence and absence of exogenouscAMP, as described above. In the presence of 10 M cAMP,there were no significant differences in maximum cAMP-activatable PKA in ventricular homogenates from sedentarycontrol, sedentary STZ-diabetic, ExT control, and ExT STZ-diabetic rat hearts (Fig. 8 A). Interestingly, in the absence ofexogenous cAMP, homogenates from sedentary STZ-diabeticrat hearts exhibited lower intrinsic PKA activity than thosefrom sedentary control animals (Fig. 8B; P 0.05). ExTblunted the extent of reduction of intrinsic PKA activity in-duced by diabetes. ExT did not alter maximum or intrinsicPKA activities in hearts from control nondiabetic rats.
Phosphorylation of CaMKII at Thr287 also was assessed asan indirect measure of CaMKII activity. As shown in Fig. 8C,homogenates from sedentary STZ-diabetic rat hearts exhibiteda 50.1 � 8.1% increased phosphorylation of CaMKII at Thr287,and this increase was blunted with ExT. Interestingly, heartsfrom ExT control animals also showed an increase in Thr287
phosphorylation.
DISCUSSION
Clinical studies have repeatedly demonstrated that ExTslows and/or delays the progression of myocardial contractilityloss induced by both type 1 and type 2 diabetes. However,molecular mechanisms underlying this beneficial effect remainincompletely characterized. In the present study a multifacetedapproach was used to reveal for the first time that ExT duringdiabetes minimizes dysregulation of RyR2 by a mechanismthat involves, at least in part, reductions in phosphorylation atSer2808 and Ser2814. The present study is unique in that ExTwas initiated 3 wk after the onset of diabetes and persisted ina graded manner for 4 wk, making it relevant to individuals
who have been diagnosed with diabetes and later advised toincorporate exercise into their daily routine. It should bementioned that the time at which ExT is initiated after the onsetof diabetes may be important if myocardial contractility loss isto be slowed. In a recent study (5), we initiated ExT 4 wk afterthe onset of diabetes and continued the ExT for 3 wk. How-ever, in that study we were unable to reverse the bradycardiaand only minimally improved cardiac output. Although thereason(s) for this is not known at this time, the increase inmalaise as diabetes progresses maybe a contributing factor. Ina more recent study, Howarth et al. (27) initiated ExT 2 moafter the onset of diabetes and also found that it was ineffectivein improving cardiac function.
Several parameters that are reliant in part on the activityof RyR2 (i.e., release of Ca2� from the SR), including�dP/dt, rate of myocyte contractility, Ca2� spark fre-quency, diastolic Ca2� release, steady-state levels of RyR2protein, Ca2� sensitivity of RyR2, and extent of RyR2phosphorylation, were assessed in this study. We also as-sessed steady-state levels of SERCA2 protein, since itsactivity dictates the Ca2� load inside the SR.
Using M-mode echocardiography, we found that 7 wk afterSTZ injections, several cardiac hemodynamic parameters werereduced. This reduction in cardiac function was not the resultof STZ toxicity per se, but rather the diabetes, given that itcould be reversed with insulin treatment (6, 7, 8, 9). Fourweeks of ExT initiated 3 wk after the onset of diabetes blunteddecreases bradycardia, percent FS, cardiac out, and QRS in-terval induced by diabetes. In this study, ExT did not signifi-cantly increase mean EF. However, since there was a trendtoward an increase, it is likely that increasing the duration ofExT would improve this hemodynamic parameter. In in vivohemodynamics studies, ExT did not increase basal �dP/dt butdid significantly increase �dP/dt in response to isoproterenolstimulation to near control values. These data are consistentwith our earlier findings showing that ExT selectively pre-serves �1-adrenoceptor expression and signaling (5). ExT alsoincreased basal �dP/dt as well as �dP/dt in response toisoproterenol. The latter finding suggests that ExT duringdiabetes is enhancing and/or increasing the activity ofSERCA2. This is especially important because diastolic heart
Fig. 8. Maximum PKA activity (A) and intrinsic or basal PKA activity (B) in homogenates prepared from sedentary control, sedentary STZ-diabetic, ExT control,and ExT STZ-diabetic rat hearts. Data are means � SE from 4 analyses. C: representative Western blots of total CaMKII (tCaMKII) and phospho-CaMKII(Thr286/287) (pCaMKII) levels. Graph at bottom shows means � SE of pCaMKII(Thr286/287) levels obtained from 5 separate preparations. *P 0.05, significantlydifferent from sedentary control.
1289EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
failure, which accounts for �50% of all heart failures, stems inpart from a loss of SERCA2 activity (17). Consistent withechocardiographic and in vivo hemodynamics data, we foundthat ExT also preserved myocyte contraction and relaxationvelocities and extent of cell shortening. To our knowledge,these data are the first to show in an experimental model thatExT initiated after the onset of diabetes blunts myocyte con-tractility loss.
Having established that ExT is effective in slowing thedevelopment of myocardial contractility, we then investigatedCa2� released from the SR. Consistent with earlier work (44,54), in the present study we found that spontaneous Ca2�
sparks, which arise from aberrant activation of RyR2, in-creased in frequency during diabetes, and this increase wasattenuated with ExT. ExT also attenuated decreases in Ca2�
transient amplitude as well as decreases in the rate of Ca2�
rise, parameters that are dependent in part on the activity ofRyR2. The activity of SERCA2 that dictates the SR Ca2� loadalso is increased with ExT (4). The amplitudes of Ca2� sparksin ExT control animals were brighter than those in sedentarycontrol animals, but their frequencies were similar. This maybe due in part to an increase in expression and/or activity ofSERCA2 and the resultant increase in SR Ca2� load. RyR2opening for a longer duration of time also may contribute.
Previously, we (44) reported that the increase in Ca2� sparkfrequency seen during diabetes maybe as a result of dissocia-tion of calstabin2 from the RyR2 complex. Consistent with thishypothesis, in the present study we found that SR membranesprepared from ExT diabetic rat hearts (which showed normal-ized Ca2� spark frequency) contained higher levels of calsta-bin2 bound to RyR2. Myocytes from ExT control hearts thatexhibited a low frequency of spontaneous Ca2� sparks also hadhigher levels of calstabin2 on RyR2.
In addition to spontaneous Ca2� release, we also investi-gated the effect of ExT on evoked Ca2� transients. Similar toour previous report (44), in the present study �50% of myo-cytes from sedentary diabetic rat hearts exhibited dyssynchro-nous Ca2� release with diastolic Ca2� release in betweenpulses. ExT initiated after the onset of diabetes also attenuateddyssynchronous and diastolic Ca2� releases. Although theincrease in diastolic Ca2� release is directly attributed todysregulation of RyR2 (gain of function), the mechanism(s)underlying dyssynchronous Ca2� release from the SR remainspoorly characterized. Previously, we postulated that this defectcould be due to alterations in the sensitivity of RyR2 to Ca2�
stimulation, since our own and the majority of studies found nochanges in the activity of L-type Ca2� current during diabetes(10, 11, 29 44). However, dyssynchronous Ca2� release alsocould arise from a disruption of the dyad junction architecture(46). In unpublished studies done in collaboration with Dr.Clara Franzini-Armstrong (University of Pennsylvania), wefound no change in the dyad junction architecture in heartsfrom control and sedentary diabetic rats.
Another major finding of the present study is that ExTduring diabetes blunts alterations in Ca2� sensitivity of RyR2induced by diabetes. Similar to earlier reports (8, 44), we foundthat at equivalent amounts, RyR2 protein from sedentary dia-betic rat hearts bound less [3H]ryanodine at peak [Ca2�]compared with RyR2 protein from sedentary control animals.Further studies are needed to determine whether this is due toa reduction in channel gating or conductance. Diabetic RyR2
also was more sensitive to Ca2� activation, and a higher[Ca2�] was needed for channel deactivation. ExT blunted theseCa2� changes. To date, mechanisms underlying altered sensi-tivity of RyR2 to Ca2� activation and/or deactivation remainpoorly defined. However, studies indicate that this could be duein part to an increase in extent of phosphorylation of RyR2 byPKA and CaMKII (25, 34, 48, 49, 50).
To address this, we used phosphospecific antibodies toassess phosphorylation of RyR2 at Ser2808 (PKA and CaMKIIsites) and Ser2814 (CaMKII site). In the present study we foundthat although total RyR2 remained unchanged, phosphoryla-tion of RyR2 at Ser2808 and Ser2814 increased significantlyduring diabetes, and these increases were attenuated with ExT.To our knowledge, these data are the first to directly show thatphosphorylation of RyR2 at Ser2814 increases during diabetesand that ExT attenuates increases in RyR2 phosphorylation atboth Ser2808 and Ser2814.
To discern which kinase is responsible for the increasedphosphorylation of RyR2 during diabetes, we assessed PKAand CaMKII activity. Consistent with an earlier study (38), wealso found increased endogenous CaMKII activity (assessedfrom phospho-Thr287). CaMKII is a multimeric holoenzymecomposed of 6–12 subunits, and the �-isoform predominates inthe heart (35). A spliced variant of this isoform, �c, is presentin the cytoplasm, and this isoform is responsible for phosphor-ylation of several Ca2� proteins, including RyR2. A rise inintracellular Ca2� causes Ca2�-bound calmodulin to bind toand activate this kinase. Binding of Ca2�-bound calmodulinresults in rapid autophosphorylation of Thr287. This autophos-phorylation renders CaMKII autonomous (i.e., active) in theabsence of Ca2�-calmodulin (35, 55). The increase in CaMKIIactivity, independently of changes in protein levels, couldresult from the increase in myocyte basal Ca2� induced bydiabetes (35). Lowering of cytosolic pH as a result of diabetes-induced ischemia also may be a contributing factor. Activationof the guanine nucleotide exchange factor activated by cAMP(Epac) as a result increased sympathetic activity, persistentstimulation of �-adrenoceptors, and elevation in myocytecAMP levels also could increase CaMKII activity (45).
Interestingly, in the present study no significant change inmaximum activatable PKA activity was detected (in the pres-ence of 10 M cAMP), but intrinsic or basal PKA activity wasreduced. The reason for this is not clear at this time, but onepossibility is that during diabetes, a greater amount of catalyticand regulatory subunits of PKA remain bound together as aresult of reduction in availability of cAMP for activation.Taken at face value, our present data suggest that the increasein phosphorylation of RyR2 at Ser2808 and Ser2814 seen duringdiabetes stems principally from an increase in CaMKII. How-ever, we have yet to investigate whether the PKA activitywithin the RyR2 macromolecular complex is higher than thecellular average during diabetes to cement this conclusion.
In the present study we found that RyR2 from ExT controlanimals also exhibited increased phosphorylation at Ser2808
and Ser2814. However, ExT RyR2 did not exhibit altered Ca2�
sensitivity as assessed using [3H]ryanodine binding assays.Myocytes from ExT control animals also did not exhibit anincrease in Ca2� spark frequency. From these data it appearsthat although increases in phosphorylation of RyR2 at Ser2808
and Ser2814 are likely to contribute to the increase in Ca2�
spark frequency (24), they are not likely to be the only
1290 EXERCISE TRAINING DURING DIABETES BLUNTS RYR2 DYSREGULATION
mechanism responsible for the altered Ca2� sensitivity ofRyR2 during diabetes. Posttranslational modifications resultingfrom increased production or availability of reactive oxygenand carbonyl species also may be contributing (8, 10, 20, 53).This is especially likely given that recent studies have shownthat ExT upregulates endogenous antioxidant defenses that arecapable of scavenging or reducing reactive oxygen and car-bonyl species (2, 12, 22). It should be pointed out that thesedata also suggest that the association of calstabin2 to RyR2may not be solely dependent on the phosphorylation state ofRyR2, at least in the context of diabetic cardiomyopathy.
In the present study and in a very recent study (44), weobserved an apparent discrepancy between the increase inCa2� spark frequency and the reduction in [3H]ryanodinebinding. One would usually expect that if there were anincrease in activity of RyR2, then there would be an increase in[3H]ryanodine binding. However, this was not the case. Wespeculated that this apparent discrepancy might be due to thepresence of two distinct populations of RyR2 in diabeticmyocytes: one population with increased responsiveness toCa2� and another population with reduced or little responsive-ness to Ca2� activation. The nonresponsive channels couldaccount for the dyssynchronous evoked Ca2� release from theSR seen in the line scan mode. Additional studies need to beconducted to further characterize Ca2�-sensitive and Ca2�-insensitive RyR2.
In conclusion, we have shown for the first time in a com-prehensive way that ExT initiated after the onset of diabetespreserves RyR2 function by reducing phosphorylation atSer2808 and Ser2814. These data also provided additional in-sights into molecular mechanisms by which ExT improvesmyocardial function during diabetes.
ACKNOWLEDGMENTS
We thank Janice Taylor of the Confocal Laser Scanning Microscope CoreFacility at the University of Nebraska Medical Center (UNMC) for providingassistance with Zeiss LSM 410 laser confocal microscope and the NebraskaResearch Initiative and the Eppley Cancer Center for support of the CoreFacility.
Use of facilities constructed by Research Facilities Improvement ProgramGrant C06 RR-17417 and the Nebraska Center for Cell Biology, (supported byEPSCoR EPS-0346476, CFD 47.076) was greatly appreciated.
GRANTS
This work was supported in part by grants from the Edna Ittner ResearchFoundation, American Diabetes Association, Dean’s Research Fund UNMC(to K. R. Bidasee), and the W. M. Keck Foundation Distinguished YoungScholars program (to X. H. T. Wehrens) and by National Institutes of HealthGrants HL-089598 (to X. H. T. Wehrens), AA-017663 (to T. A. Wyatt),NS-39751 (to K. P. Patel), HL-066446 (to G. J. Rozanski), and HL-085061 (toK. R. Bidasee).
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