Interval Training Normalizes Cardiomyocyte Function, Diastolic Ca2+ Control, and SR Ca2+ Release Synchronicity in a Mouse Model of Diabetic Cardiomyopathy
Rationale: In the present study we explored the mechanisms behind excitation–contraction (EC) coupling defects in cardiomyocytes from mice with type-2 diabetes (db/db).
Objective: We determined whether 13 weeks of aerobic interval training could restore cardiomyocyte Ca2+ cycling and EC coupling.
Methods and Results: Reduced contractility in cardiomyocytes isolated from sedentary db/db was associated with increased diastolic sarcoplasmic reticulum (SR)-Ca2+ leak, reduced synchrony of Ca2+ release, reduced transverse (T)-tubule density, and lower peak systolic and diastolic Ca2+ and caffeine-induced Ca2+ release. Additionally, the rate of SR Ca2+ ATPase–mediated Ca2+ uptake during diastole was reduced, whereas a faster recovery from caffeine-induced Ca2+ release indicated increased Na+/Ca2+-exchanger activity. The increased SR-Ca2+ leak was attributed to increased Ca2+-calmodulin–dependent protein kinase (CaMKIIδ) phosphorylation, supported by the normalization of SR-Ca2+ leak on inhibition of CaMKIIδ (AIP). Exercise training restored contractile function associated with restored SR Ca2+ release synchronicity, T-tubule density, twitch Ca2+ amplitude, SR Ca2+ ATPase and Na+/Ca2+-exchanger activities, and SR-Ca2+ leak. The latter was associated with reduced phosphorylation of cytosolic CaMKIIδ. Despite normal contractile function and Ca2+ handling after the training period, phospholamban was hyperphosphorylated at Serine-16. Protein kinase A inhibition (H-89) in cardiomyocytes from the exercised db/db group abolished the differences in SR-Ca2+ load when compared with the sedentary db/db mice. EC coupling changes were observed without changes in serum insulin or glucose levels, suggesting that the exercise training–induced effects are not via normalization of the diabetic condition.
Conclusions: These data demonstrate that aerobic interval training almost completely restored the contractile function of the diabetic cardiomyocyte to levels close to sedentary wild type.
- diabetes mellitus
- exercise training
- Ca2+-calmodulin–dependent protein kinase
- ryanodine receptor and calcium handling
Diabetes mellitus (type 2 diabetes) is estimated to reach pandemic levels within the next 2 decades.1 This has severe implications, because cardiovascular mortality is ≈2- to 4-fold higher in diabetic compared to nondiabetic patients2 and accounts for ≈80% of the mortality in type 2 diabetes,3 of which ≈50% die of sudden cardiac death.4 Furthermore, diabetics are 2.5 times more likely to develop congestive heart failure compared to nondiabetics.5
The db/db diabetic mouse model develops cardiomyopathy in a similar manner as type 2 diabetes in humans,6 and presents with reduced whole-heart7 and isolated cardiomyocyte8 excitation–contraction (EC) coupling function. This can partly be explained by a reduced L-type Ca2+ channel activity and increased Na2+/Ca2+ exchanger (NCX) and depressed Ca2+ handling by the sarcoplasmic reticulum (SR).8,9 Furthermore, increased SR Ca2+ leak in db/db mice8 can further reduce SR Ca2+ content. Recently, arrhythmias have been linked to increased diastolic Ca2+ leak via the SR release channels, the ryanodine receptor 2 (RyR2), causing delayed afterdepolarizations.10 In failing hearts, phosphorylation of RyR2 by the Ca2+-calmodulin–dependent protein kinase IIδ (CaMKIIδ) or the protein kinase A (PKA) is thought to sensitize RyR2 to Ca2+ and thus increase its open probability.11,12 In contrast, exercise training in healthy mice increases the level of phosphorylated cytosolic CaMKIIδ and in so doing increases CaMKIIδ activity. Under these circumstances, increased phosphorylation of CaMKIIδ was associated with an increased cardiac performance.13
Alongside increased SR Ca2+ leak, reduced transverse (T)-tubule structure leading to less synchronous SR Ca2+ release contributes further to the depressed EC coupling in models of cardiac dysfunction.14 The mechanism for increased SR Ca2+ leak, and whether T-tubule structure in diabetic cardiomyopathy is conserved, has currently not been studied.
In the present study, we explored the mechanisms behind the impaired cardiomyocyte function and increased SR Ca2+ leak in db/db cardiomyocytes, and then reexamined the same parameters in the db/db mice after an aerobic interval exercise training program. Because the activities of both CaMKIIδ and PKA are associated with both pathological and physiological remodeling, we also investigated the contributions of CaMKIIδ and PKA for the observed exercise training-induced changes.
For a detailed description, see the data supplement (available online at http://circres.ahajournals.org).
Mouse Model of Diabetes and Exercise Training
The db/db mice model has been proven to be a suitable model to study the consequences of diabetes on the heart. Here we studied the male diabetic (BKS.Cg-m +/+ Lepdb/Bom Tac; 20 exercised and 20 sedentary mice) and sedentary (n=23) and exercise trained (n=6) nondiabetic healthy heterozygote (BKS.Cg-m +/+ Lepdb/+ lean); all age-matched (7 weeks at study start). To determine maximal oxygen uptake (VO2max), mice ran until exhaustion on a customized treadmill in a metabolic chamber, and high-intensity aerobic interval training was performed as uphill running, alternating between 4 minutes at 85% to 90% of VO2max and 2 minutes at 50% of VO2max for 80 minutes/day, 5 days/wk, for 13 weeks.15 We and others have previously demonstrated the efficacy and relevance of this exercise regime by both clinical trials and experimental studies (eg, see Tjonna et al16). The Norwegian council for Animal Research approved the study, which was in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1996).
Cardiomyocyte Isolation and Ca2+ Measurements
Left ventricular myocytes were isolated as previously described.15 Fura-2/AM-loaded cardiomyocytes were stimulated by bipolar electric pulses for Ca2+ handling measurements including SR Ca2+ leak. CaMKII inhibitor and PKA inhibitor were used to determine the influence of the 2 kinases. Contractility was recorded by video-based sarcomere spacing.
Confocal Imaging of Ca2+ Waves, Ca2+ Release Synchrony, and T-Tubules
Cardiomyocytes loaded with Fluo-3/AM were used to count Ca2+ waves and determine Ca2+ release synchrony. Quiescent nonperfused cardiomyocytes loaded with the membrane specific Di-8-ANEPPS were confocal Z-stack scanned. The relative density of T-tubules normalized to cell size was obtained from 5 images per cell captured from the middle of each cell.
Western Blot Analyses and Real-Time Quantitative RT-PCR
Western Blot and real-time quantitative RT-PCR analysis were performed using standardized protocols and normalized to housekeeping proteins and genes. For a detailed description see online supplement.
Data are shown as mean±SD. One-way ANOVA with Bonferroni posthoc test adjusted for multiple comparisons was used to identify the statistical differences between the groups and Mann-Whitney U was used when appropriate. P<0.05 was considered statistically significant.
Aerobic Capacity and Echocardiography
Exercise training improved aerobic capacity, and exercised db/db mice had a 13% higher VO2max than sedentary db/db and WT mice (Figure 1A). Improved aerobic fitness was also reflected by increased maximal running speed to a level above that of sedentary mice (0.23 versus 0.12 m · s−1, respectively; P<0.001). Exercise training did not significantly change the body weight (Figure 1B). Left ventricular dysfunction in sedentary db/db mice was confirmed by reduced fractional shortening and stroke volume by high-resolution echocardiography, whereas endurance training improved both parameters to wild-type levels (Figure 1C and 1D).
Exercise Training in WT Animals
To compare the functional response to exercise training in diabetic mice to normal mice, we also exercise trained WT mice. Overall, results from exercised WT were superior to those observed in sedentary and exercised db/db and sedentary WT mice (Figures 1 through 6⇓⇓⇓⇓⇓; Table).
Free Fatty Acids, Triglycerides, Insulin, and Blood Glucose
Sedentary db/db mice had higher plasma levels of free fatty acids (648±90 versus 263±70 mmol/L, P<0.05) and similar triglycerides (0.74±0.06 versus 0.68±0.07 mmol/L, NS) when compared to sedentary WT mice. Exercise training reduced free fatty acids by ≈16% (to 544±66 mmol/L, P<0.05) and triglycerides by ≈23% (to 0.56±0.05 mmol/L, P<0.05). Plasma glucose was higher in db/db mice (20.1±0.9 versus 10.3±0.8 mmol/L, P<0.01) than in sedentary WT, and exercise training did not lower it significantly (17.0±1.2 mmol/L, P=0.19). As expected, insulin was substantially higher in sedentary db/db mice (6.65±0.92 μg/L) versus sedentary WT (0.98±0.01 μg/L), with no change observed after the training period (6.89±1.22 μg/L).
Cardiac mRNA levels of peroxisome proliferator–activated receptor γ coactivator 1α (PGC-1α) is a critical factor associated with the activation of metabolic genes required for substrate use and mitochondrial biogenesis. These levels were similar in sedentary db/db and sedentary WT (0.97±0.11 arbitrary units) and exercise training did not change PGC-1α (0.89±0.12).
Fractional Shortening and Ca2+ Cycling
Fractional shortening was impaired in sedentary db/db mice, but recovered to a level comparable to sedentary WT mice after the exercise program (Figure 2A and 2C). In line with this, we observed lower twitch Ca2+ release in sedentary but not in exercised db/db mice compared to sedentary WT mice (Figure 2B and 2D). Sedentary db/db mice had lower diastolic and systolic Ca2+ levels, as well as reduced amplitude of the Ca2+ transient, compared to sedentary WT. After exercise training, no differences were observed between db/db and sedentary WT mice (Figure 2B and 2D). Lower SR Ca2+ load in sedentary db/db mice was confirmed by lower caffeine-induced Ca2+ release (Table). Exercise training increased SR Ca2+ load, demonstrated by increased caffeine-induced Ca2+ release; however, it did not reach sedentary WT levels (Table).
Diastolic function, measured as time to 50% relengthening, was impaired in sedentary db/db mice compared to sedentary WT mice, but was restored after exercise training (Figure 2E). The same pattern of change was observed for time to 50% Ca2+ transient decay (Figure 2F). Sarcoplasmic reticulum Ca2+ ATPase (SERCA) is the main contributor to removal of cytosolic Ca2+ during diastole, and also indirectly affects NCX by virtue of controlling [Ca2+]i. Rate constants of Ca2+ decay indicated that sedentary db/db mice had a ≈34% suppression of SERCA2a function and 59% increased NCX function compared to sedentary WT mice. Exercise training restored the functions of SERCA2a and NCX to levels comparable to sedentary WT mice (Figure 3B through 3D). This suggests that exercise training normalizes diastolic function in db/db mice by shifting the control of diastolic Ca2+ to SERCA2a and thus increasing the rate of Ca2+ removal. This also increased the SR Ca2+ load, which may contribute to improved systolic function.
SR Ca2+ Leak
Measuring [Ca2+]i in quiescent cardiomyocytes over a prolonged period of time (1 minute) with and without tetracaine provides a quantitative assessment of SR (RyR2) Ca2+ leak (Figure 4A). After normalizing for differences in SR Ca2+ content, we observed an increased SR Ca2+ leak in sedentary db/db mice compared to sedentary WT mice, whereas exercise training normalized SR Ca2+ leak in db/db mice to levels comparable to sedentary WT mice. 13% of the total SR Ca2+ content leaked during this period in sedentary db/db mice, versus 3% to 4% in both exercised db/db and sedentary WT mice (Figure 4B). This was also true when expressed as absolute Ca2+ leak (not normalized for differences in SR Ca2+ content, data not shown). In line with this, the frequency of nonstimulated Ca2+ waves was higher in sedentary db/db mice compared to exercised db/db and sedentary WT mice (Figure 5A and 5B). To further elucidate the mechanism behind increased SR Ca2+ leak, we inhibited CaMKIIδ with AIP and PKA with H-89. SR Ca2+ leak was unaffected by PKA inhibition, whereas CaMKIIδ inhibition reduced Ca2+ leak in sedentary db/db mice to levels comparable to exercised db/db and sedentary WT mice (Figure 4B and 4C).
Reduced Synchrony of Ca2+ Release, T-Tubules, and Cardiomyocyte Size
To further examine how diabetes may affect Ca2+ handling and contractility, we measured the synchrony of Ca2+ release during twitch stimulations. Compared to sedentary WT mice, Ca2+ release along the cardiomyocyte length was less synchronous in sedentary db/db mice, but exercise training reversed this to levels comparable to sedentary WT mice (Figure 6A and 6C). Synchrony of Ca2+ release is closely linked to the density and organization of T-tubules in the cardiomyocyte.14 In line with this, we observed a reduced T-tubule density in sedentary db/db mice compared to sedentary WT mice, whereas exercise training restored the density (Figure 6B and 6D). Cardiomyocytes from sedentary db/db mice had an approximately 63% larger volume than cardiomyocytes from sedentary WT mice (P<0.01). Exercise training reduced cell volume to a level comparable to sedentary WT (Figure 6E).
Protein Expression and Phosphorylation Status
SERCA2a expression was reduced in sedentary db/db mice compared to sedentary WT mice, but exercise training normalized this (Figure 7A). Total phospholamban (PLN) expression levels did not differ between groups (Figure 7B). Phosphorylation of PLN at the CaMKIIδ site (Threonine-17) was increased in sedentary db/db mice compared to sedentary WT mice (Figure 7C), whereas PLN phosphorylation at the PKA site (Serine-16) was similar between sedentary db/db and sedentary WT mice (Figure 7D). Exercise training strongly increased PLN phosphorylation at Serine-16 (compared to sedentary db/db and sedentary WT mice) but reduced PLN phosphorylation at Threonine-17 (Figure 7C and 7D). Thus, exercise training normalized the phosphorylation status of PLN Threonine-17 but not Serine-16. Finally, expression levels of CaMKIIδ did not differ between the groups (Figure 7E), but phosphorylation of CaMKIIδ was strongly increased in sedentary db/db mice, whereas exercise training reduced this to sedentary WT levels (Figure 7F). Expression levels of RyR (1.17±0.06, 1.30±0.05 and 1.20±0.05 in sedentary db/db, exercised db/db and sedentary WT, respectively) and phosphorylation of RyR at the PKA and CaMKIIδ site Serine-2808 (Figure 7G) were similar between groups, whereas phosphorylation at the CaMKIIδ-specific site Serine-2814 was higher in sedentary and normalized in trained db/db, when compared to WT controls (Figure 7H).
Effects of CaMKII and PKA Inhibitors
Overall, inhibition of PKA (with H89) had more profound effects than inhibition of CaMKIIδ (with AIP). Across all the groups, PKA inhibition induced the greatest reductions in fractional shortening, diastolic function, twitch Ca2+ amplitude and decay, and SR Ca2+ content (Table). Interestingly, inhibition of PKA in exercise trained db/db mice resulted in fractional shortening, diastolic function, twitch Ca2+ amplitude and decay, and SR Ca2+ content being similar to sedentary db/db (group differences ns, Table). Fractional Ca2+ release was enhanced in sedentary db/db mice compared to sedentary WT mice, but exercise training normalized this (P<0.05, Table). Adding AIP to the db/db sedentary cardiomyocytes reduced fractional Ca2+ release to levels comparable to exercised db/db and sedentary WT (Table).
In the present study, we investigated subcellular mechanisms of dysfunction in diabetes-induced cardiomyopathic hearts, and the potential of regular exercise training for correcting the dysfunction. Dysfunction and cardiomyopathy in the present diabetes model was evidenced by reduced fractional shortening and stroke volume in vivo, reduced cell contractility and Ca2+ handling, and induction of pathological hypertrophy, in concordance with previous findings.8,9 Moreover, 2 novel mechanisms of dysfunction in the diabetic cardiomyocyte were identified in the present study, (1) asynchronous EC coupling that was associated with reduced density of T-tubules and (2) increase of diastolic SR Ca2+ leak that was associated with increased phosphorylation of cytosolic CaMKIIδ. Both are linked to reduced contractility and Ca2+ handling. High-intensity exercise training restored synchrony of EC coupling and T-tubule density and reduced SR Ca2+ leak to levels comparable to sedentary WT mice. These effects also coincided with exercise-induced restoration of contractility and Ca2+ handling to normal levels. In contrast, exercise training did not alter serum glucose and insulin concentrations, in line with a previous study.17 This suggests that normalized insulin and glucose transport cannot account for the restoration of the contractile function. Instead, other factors intrinsic to the myocardium explain the exercise training–induced effects. Previous studies suggest that aspects of the diabetic phenotype may be attributable to the disruption of a number of intracellular pathways, including those linked to glucose and insulin signaling. One possible candidate, namely mitochondrial biogenesis (PGC-1α), was ruled out by the current study. Furthermore, previous studies have shown that normalization of glucose and palmitate oxidation by high glucose and insulin treatment did not normalize cardiac function.18 Other possible pathways, including endogenous reactive oxygen species scavengers,19 advanced glycation end products,20 O-linked N-acetylglucosamine,21 and Akt signaling,22 have been suggested; clearly further studies are required to uncover the key pathways altered by exercise.
Increased Diastolic SR Ca2+ Leak and Spontaneous Waves in Diabetes
Increased SR Ca2+ leak and increased frequency of spontaneous Ca2+ waves during diastole in quiescent cardiomyocytes were observed. These results are in line with previous reports of reduced RyR2 stability and subsequently increased SR Ca2+ leak in this model of diabetes,8 as well as being consistent with increased RyR2 activity increasing the frequency of Ca2+ waves.23 The increased diastolic SR Ca2+ leak appeared to be caused by increased CaMKIIδ activity and not PKA activity. This observation was supported by differences in phosphorylation status of RyR at Serine-2814 (CaMKIIδ phosphorylation site on RyR), but similar phosphorylation at Serine-2808 (PKA and CaMKIIδ). Despite the increased diastolic Ca2+ leak, the diabetic cardiomyocytes had lower diastolic concentrations of Ca2+ compared to wild-type controls. This can be explained by the increased activity of NCX reported in this study, though the decreased SERCA2a function may result in diastolic Ca2+ not being reduced at higher stimulation frequencies (>3 Hz), because reduced SERCA2a tends to raise end-diastolic Ca2+.
Asynchrony of Intracellular Ca2+ Release and Reduced T-Tubule Density in Diabetes
Cardiomyocytes isolated from sedentary db/db mice had a reduced synchrony of twitch-stimulated intracellular Ca2+ release. This was associated with a reduced density of the T-tubule network in the cell. Reduced synchrony of Ca2+ release and EC coupling has been observed regularly after reduced T-tubule density.14 Thus, these reports suggest that the reduced T-tubule density contributes to reduce the synchrony of Ca2+ release and EC coupling. Further support for this hypothesis comes from the observation that the response time from stimulation to Ca2+ release is longer in cardiomyocytes from sedentary db/db mice compared to sedentary WT. The reduced T-tubule density suggests a disrupted spacing between L-type Ca2+ channels and RyR2 such that EC coupling becomes inefficient. To our knowledge, the current exercise training program is the first recorded intervention to restore T-tubule density and restore the synchrony of Ca2+ release and EC coupling. Whether restoration of T-tubule density after exercise training in db/db mice is a function of reduced cell size or increased amount of T-tubules per se is presently not fully understood. T-tubule density remained unchanged when cell size increased in exercise trained WT mice. This indicates that cell size and T-tubule density can vary independently.
Reduced Cardiomyocyte Inotropy in Diabetes
The reduced inotropy in diabetic cardiomyocytes, observed as reduced amplitudes and decay rates of the fractional shortening and the Ca2+ transient, is most likely explained by several factors. The primary cause is reduced SR Ca2+ content as this reduces the amplitude of the Ca2+ transient.23 The reduced SR Ca2+ loading may be caused by the increased NCX activity that competes with SERCA2a to remove intracellular Ca2+ during diastole. In addition, Ca2+ uptake rate via SERCA2a is also reduced in this model; the combination of the two would favor Ca2+ efflux via NCX rather than reuptake into the SR. The increased diastolic SR Ca2+ leak is an additional factor contributing to a reduction in SR Ca2+ loading. Reduced synchrony of twitch-stimulated Ca2+ release would also impair EC coupling and reduce the inotropy. This is associated with, and may be caused by, reduced T-tubule density observed in this model. The relative contributions of increased NCX, increased SR Ca2+ leak, reduced SERCA2a Ca2+ uptake, and reduced T-tubule density cannot easily be assessed, but all tend to reduce Ca2+ release during EC coupling and thereby impair cellular contraction.
The Potential of Exercise Training to Reverse Mechanical Cellular Dysfunction
Here, we show for the first time that exercise training normalizes or reduces the dysfunction of both systolic and diastolic cellular parameters, such as T-tubule density, synchrony of SR Ca2+ release, diastolic SR Ca2+ leak, and the frequency of spontaneous Ca2+ waves, in cardiomyocytes from diabetic cardiomyopathy hearts. Also, SERCA2a uptake and NCX activity were all returned to normal levels. Finally, exercise training also induced reverse remodeling of diabetic cardiomyocytes, evidenced by reduced cellular dimensions as well as improved fractional shortening and stroke volume in vivo in the exercise-trained group.
The observations in this study are consistent with exercise training improving cardiomyocyte Ca2+ handling and contractility in postmyocardial infarction heart failure,24 as well as exercise training improving whole-heart function in diabetes.25 Thus, despite the pathological remodeling and contractile dysfunction, cardiomyocytes maintain the ability to respond to exercise training.13,15
Involvement of CaMKII and PKA
As discussed above, the cellular studies suggest that the increased SR Ca2+ leak in diabetic cardiomyocytes was caused by the increased CaMKIIδ activity. This is in line with the observation that CaMKIIδ was constitutively hyperphosphorylated in these cardiomyocytes. The reason for the hyperphosphorylation is unknown, but it appears despite reduced diastolic Ca2+ levels. Exercise training was able to fully reverse the SR Ca2+ leak and the constitutive hyperphosphorylation of CaMKIIδ. This suggests that exercise training–induced dephosphorylation of CaMKIIδ abolished the abnormally high SR Ca2+ leak in the diabetic model. This is consistent with the data from comparable heart failure models that link increased CaMKIIδ with higher SR Ca2+ leak, and that show inhibition of CaMKIIδ, but not PKA, abolishes the leak.11,26 db/db mice are more prone to arrhythmias,27 but the underlying mechanisms are unknown. Increased SR Ca2+ leak and increased NCX activity have been suggested as changes that would promote arrhythmias in whole hearts10; exercise training could therefore be an effective treatment for arrhythmias in diabetes.
In contrast to regulation of SR Ca2+ leak, the inotropy state of diabetic cardiomyocytes was sensitive to inhibition of both PKA and CaMKIIδ. This is also in contrast to previous studies of other forms of heart disease11,26 or exercise training in healthy normal mice,13 which have suggested CaMKIIδ and not PKA as the main kinase that chronically mediates inotropy. The reason for this paradox is unknown, but it suggests that exercise training may correct diabetes-induced cardiac cell abnormalities by both CaMKIIδ- and PKA-mediated effects. However, PLN was chronically hyperphosphorylated at the PKA-targeted Serine-16 residue in the diabetic model, suggesting that the myocardium may have a limited response to β-adrenergic stimulation.
A program of exercise training reversed the contractile abnormalities associated with diabetic cardiomyopathy and restored the cardiomyocyte Ca2+ handling and contractility to levels comparable to sedentary WT, mainly by CaMKIIδ dephosphorylation and compensatory PKA-dependent phosphorylation. This was achieved without normalizing serum insulin or glucose levels. One explanation is that exercise training has stimulated a different trophic pathway from that activated by diabetes. The consequence of the change in expression of a range of proteins that alter the cellular phenotype and as a consequence CaMKIIδ activity is reduced. Future studies will examine the upstream signaling pathways responsible for exercise-induced changes to determine whether these pathways can be accessed as a therapeutic strategy.
Sources of Funding
The present study was supported by grants from the Norwegian Council of Cardiovascular Disease, the Norwegian Research Council (Funding for Outstanding Young Investigators, UW), Simon Fougner Hartmanns Family Foundation, British Heart Foundation, Funds for Cardiovascular and Medical Research at St Olav’s University Hospital, Trondheim, and the Torstein Erbo’s Foundation, Trondheim. The funding organizations had no role in the design and conduct of the study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript.
Original received November 13, 2008; first resubmission received March 23, 2009; second resubmission received April 24, 2009; revised resubmission received July 14, 2009; accepted July 31, 2009.
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