Sarco/endoplasmic reticulum calcium ATPase activity is unchanged despite increased myofilament calcium sensitivity in Zucker type 2 diabetic fatty rat heart

Systolic and diastolic dysfunction in diabetes have frequently been associated with abnormal calcium (Ca2+) regulation. However, there is emerging evidence that Ca2+ mishandling alone is insufficient to fully explain diabetic heart dysfunction, with focus shifting to the properties of the myofilament proteins. Our aim was to examine the effects of diabetes on myofilament Ca2+ sensitivity and Ca2+ handling in left ventricular tissues isolated from the same type 2 diabetic rat hearts. We measured the force-pCa relationship in skinned left ventricular cardiomyocytes isolated from 20-week-old type 2 diabetic and non-diabetic rats. Myofilament Ca2+ sensitivity was greater in the diabetic relative to non-diabetic cardiomyocytes, and this corresponded with lower phosphorylation of cardiac troponin I (cTnI) at ser23/24 in the diabetic left ventricular tissues. Protein expression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), phosphorylation of phospholamban (PLB) at Ser16, and SERCA/PLB ratio were lower in the diabetic left ventricular tissues. However, the maximum SERCA Ca2+ uptake rate was not different between the diabetic and non-diabetic myocardium. Our data suggest that impaired contractility in the diabetic heart is not caused by SERCA Ca2+ mishandling. This study highlights the important role of the cardiac myofilament and provides new insight on the pathophysiology of diabetic heart dysfunction.

www.nature.com/scientificreports/ was increased in type 2 diabetic cp/cp rat hearts, but intracellular Ca 2+ concentration was not different at basal conditions 17 . Daniels et al. also recently found that cardiac function could be restored in diabetic rat trabeculae by kinase inhibition without any change in Ca 2+ flux 20 . These findings suggest that SERCA alone does not regulate the intracellular Ca 2+ state, and that altered intracellular Ca 2+ regulation is not the only factor causing contractile dysfunction in diabetic hearts. An alternative explanation for impaired contractility is that the Ca 2+ sensitivity of cardiac myofilaments is altered in the type 2 diabetic heart. The Ca 2+ sensitivity of the myofilament is affected by transient posttranslational modifications of myofilament proteins 21 , particularly cardiac troponin I (cTnI) 22,23 . In both type 1 and type 2 diabetic animal models, myofilament Ca 2+ sensitivity of cardiomyocytes has been reported as unaltered 6,24 , increased 25 or decreased 26 . The obese Zucker diabetic fatty (ZDF) rat is a type 2 diabetic model with well-established cardiac inotropic and lusitropic dysfunction 3,6,27 . Our group recently found that myofilament Ca 2+ sensitivity was increased in 20-week-old type 2 diabetic ZDF rat cardiomyocytes, and this was associated with reduced phosphorylation of cTnI at Ser23/24 28 . Interestingly, increased myofilament Ca 2+ sensitivity would be expected to enhance cardiac contractility by increasing the affinity of thick and thin filament interaction 29 . Therefore, it is unclear why contractile dysfunction still existed in these hearts. One possibility is that deranged SERCA activity reduces intracellular Ca 2+ concentrations. Reduced SERCA Ca 2+ uptake has been associated with reduced systole Ca 2+ transient amplitude and impaired cardiac contractility in type 2 diabetic mouse heart 11 . Reduced SERCA-mediated Ca 2+ uptake activity may decrease sarcoplasmic reticulum Ca 2+ content, hence less intracellular Ca 2+ would be available for the subsequent contractions, and this may explain systolic dysfunction in the diabetic heart.
As our group has recently found that myofilament Ca 2+ sensitivity is increased in type 2 diabetic heart 28 which is not normally associated with contractile dysfunction, we therefore hypothesised that SERCA expression and activity would be reduced in 20-week-old type 2 diabetic rat heart.

Discussion
The obese diabetic ZDF rat heart is characterised by depressed contractility but increased myofilament Ca 2+ sensitivity 28 . This implies that reduced Ca 2+ availability exists during systole in order to account for reduced force generation. In this context, we hypothesised that the activity of SERCA, the protein most responsible for the intracellular Ca 2+ flux, would be lower in the obese diabetic ZDF rat left ventricle. However, SERCA activity was not different between groups, despite the fact that the SERCA/PLB ratio was lower in the diabetic hearts. Similarly, the expression of NCX, which extrudes Ca 2+ from the cardiomyocyte, was not different between groups.  www.nature.com/scientificreports/ As previously shown 28 , we found that myofilament Ca 2+ sensitivity was greater, and that this augmentation was associated with lower phosphorylation of cTnI at Ser23/24 in the diabetic hearts compared to the non-diabetic hearts. Therefore, while left ventricular contractility is reduced in this diabetic heart model, our findings suggest this dysfunction is not caused by changes in SERCA activity. Increased myofilament Ca 2+ sensitivity in the obese diabetic ZDF rat cardiomyocyte suggests that less Ca 2+ would be required to elicit a given contractile force than in the non-diabetic cardiomyocytes. Numerous isolated heart experiments have shown that left ventricular force is reduced in this model 3,27 ; which when combined with increased Ca 2+ sensitivity, implies that the intracellular Ca 2+ concentration in diabetic cardiomyocytes is reduced; even in the absence of any difference in SERCA activity. Indeed, Belke et al. found that intracellular Ca 2+ concentration during systole and diastole was significantly lower in type 2 diabetic db/db mouse isolated myocytes 9 , which they attributed to decreased Ca 2+ handling by the sarcoplasmic reticulum. However, Zhang et al. argued that reduced expression of SERCA could explain prolonged action potentials and slower Ca 2+ transients, but not contractile deficits; which they attributed to remodelling of the myofilament, specifically a reduction in actin content 18 . Others have reported that reduced NCX activity impairs contractile function in type 1 diabetic rat models 30,31 . We did not quantify intracellular Ca 2+ transient amplitude in our experiments, but found no evidence to suggest that deranged Ca 2+ handling by either SERCA or NCX was associated with poor contractility in our experiment. Nonetheless, the combination of decreased contractility of isolated hearts and increased myofilament Ca 2+ sensitivity support the idea that intracellular Ca 2+ concentration may have been lower in the diabetic myocardium.
Although the protein expression of SERCA and PLB have been used as indicators of SERCA activity, our data, and others 12,32 suggest that protein levels do not necessarily reflect the intrinsic SERCA activity. We found that SERCA/PLB ratio and phosphorylated PLB at Ser16 were lower in the diabetic hearts, without any difference in the maximum SERCA Ca 2+ uptake rate between groups. The discrepancy between SERCA expression and activity may be explained by the 'coupling' between SERCA and PLB. Several studies have shown that only a fraction of SERCA is functionally regulated by PLB in the heart 33,34 . Brittsan et al. found that SERCA Ca 2+ uptake rate was proportionately reduced with increasing PLB expression in transgenic mouse model, and only ~ 40% of SERCA was under the inhibitory regulation of PLB in murine hearts under basal conditions 34 . This suggests that the 'coupling' between SERCA and PLB may not be affected by diabetes in the obese diabetic ZDF rat myocardium  www.nature.com/scientificreports/ and this may explain why SERCA Ca 2+ uptake activity was unaltered despite a lower SERCA/PLB ratio in the diabetic left ventricular tissues. Our finding that the diabetic ZDF rat heart had greater myofilament Ca 2+ sensitivity and lower phosphorylation of cTnI at Ser23/24 28 reinforces the inverse association between myofilament Ca 2+ sensitivity and phosphorylation of cTnI at Ser23/24 described in heart disease 21,35 . Similarly, our data show that phosphorylation of PLB at Ser16 was lower in the diabetic rat left ventricular tissues. Phosphorylation of cTnI at Ser23/24 and PLB at Ser16 are commonly recognised as PKA-dependent phosphorylation sites mediated by the cyclic adenosine monophosphate (cAMP) pathway 21,36 . Bockus and Humphries found that inotropic and lusitropic responses were impaired in type 1 diabetic mice following acute activation of PKA 37 . Thaung et al. also found that the expression of G s protein, which increases cAMP level during β-adrenergic stimulation, was reduced by 28% in this type 2 diabetic ZDF rat model 27 . Therefore, our data suggest that cAMP/PKA signalling may be impaired in the diabetic heart. Interestingly, Daniels et al. found that contractility and relaxation were restored in diabetic rat trabeculae without any change in sarcoplasmic reticulum Ca 2+ flux when Ca 2+ /calmodulin dependent kinase II (CaMKII) was inhibited 20 . Unfortunately, the authors did not determine whether this effect was associated with alterations in the phosphorylation of cTnI or other myofilament proteins known to alter Ca 2+ sensitivity.
Though not the focus of our study, the functional consequences of increased myofilament Ca 2+ sensitivity may be involved in diabetic diastolic dysfunction. Increased baseline systole 38 , prolonged systolic duration 39 and slower diastolic relaxation 13 have been identified as early manifestations of diabetes, which precede cardiovascular disease. The greater myofilament Ca 2+ sensitivity in the diabetic rat hearts in this study may suggest stronger binding of Ca 2+ ions to cardiac troponin C (cTnC), which would delay or reduce the rate of Ca 2+ dissociation from cTnC 40,41 . This increased affinity of Ca 2+ for the troponin complex would prolong thick and thin filament  www.nature.com/scientificreports/ interactions, thereby 'delaying' the onset of relaxation, leading to incomplete cardiac relaxation as often described in the diabetic heart 27 . Our data may indicate that diabetes impacts the force generating components of the myofilament itself. It is possible that diabetes reduced cardiomyocyte force generation by interfering with the Ca 2+ -myofilament interaction. We found that skinned diabetic cardiomyocytes tended to produce less maximal force (~ 25%, P = 0.06) when bathed in supra-physiological Ca 2+ (pCa 4.5). In such conditions, it is assumed that all thick and thin filament cross-bridges are activated, and any difference in force is explained by differences in the quantity of force producing cross-bridges per unit area. Jweied et al. similarly found that maximal force was 29% lower in diabetic human skinned cardiomyocytes (P = 0.08 vs. non-diabetic controls) 42 and van den Brom et al. found that maximal force was 68% lower in 14-week-old type 2 diabetic ZDF rats with impaired cardiac function 6 . Zhang et al. found that the area of the actin filament was 8% lower in streptozotocin-induced diabetic rat myofilaments, and that the sarcomeric organization of actin was disorganized compared to non-diabetic myofilaments 18 . A reduction in the content or quality of force generating proteins in the diabetic left ventricular cardiomyocyte would explain the consistent finding that maximal force is reduced in isolated heart experiments in this model 3,6,27 . It is also possible that the increase in myofilament Ca 2+ sensitivity would compensate for this loss of generated force by eliciting greater contractile activation at lower intracellular Ca 2+ concentrations.
Previous studies by our group have consistently shown that type 2 diabetic ZDF rats have impaired cardiac contractility 20,27 and coincide with increased myofilament Ca 2+ sensitivity 28 . SERCA is the primary Ca 2+ pump in cardiomyocytes; however, the lack of change in SERCA activity may indicate that an alternative Ca 2+ transporter is altered by diabetes. Several studies have suggested that NCX activity is increased in diabetic cardiomyocytes 8,11 , and may 'compete' with SERCA to remove Ca 2+ from the cytosol. We did not measure NCX activity in the present study. However, NCX expression was not different between the diabetic and non-diabetic myocardium, which may indicate a normal Ca 2+ efflux under basal condition. Similarly, Belke et al. found that NCX activity was not different between type 2 diabetic and non-diabetic hearts 9 . A more exhaustive comparison of the various Ca 2+ ion channels (i.e. L-type, NCX, ryanodine receptors, SERCA), and the dynamic interactions between Ca 2+ and the myofilaments may be necessary to better understand the mechanism of diabetic heart dysfunction.
Our data show that the type 2 diabetic left ventricular cardiomyocytes have greater myofilament Ca 2+ sensitivity and lower phosphorylation of cTnI at Ser23/24. SERCA/PLB ratio was reduced, however, SERCA Ca 2+ uptake activity was unaltered in the diabetic hearts, suggesting that impaired contractility may be associated with changes in intracellular Ca 2+ concentration independent of SERCA. These findings suggest an important role for myofilaments and generate new questions regarding the pathophysiological mechanism in diabetic heart dysfunction.

Methods
Animals. Experiments were conducted on 20-week-old male type 2 diabetic Zucker Diabetic Fatty (ZDF fa/fa ; n = 5) rats and their non-diabetic (homozygous ZDF +/+ or heterozygous ZDF +/-; n = 5) littermates. Animals were obtained from an in-house colony, with the original breeding pairs obtained from Charles River Laboratories (Wilmington, MA, USA). Animals were housed in groups of 2 per cage with a standard 12-h light-dark cycle (0700 -1900) and stable temperature of 21 ± 1 °C. Animals were fed with rat chow Purina 5008 (LABDIET) and had access to food and water ad libitum.
At 20 weeks of age, the animals were weighed and deeply anaesthetised with 60 mg/kg sodium pentobarbital via intraperitoneal injection. When the pedal withdrawal reflex was absent, the chest was cut open, the heart was quickly excised and rinsed in ice-cold Krebs-Henseleit buffer. The left ventricle was cut into several pieces, flash frozen in liquid nitrogen and stored at -80 °C. A thorax blood plasma glucose test was performed using a glucometer and test strips (ACCU-CHEK Performa, Roche) to confirm diabetic status.
Cardiomyocyte force-pCa measurements. Force measurements were performed on isolated skinned cardiomyocyte preparations at 15 °C as described by Diffee and Nagle 43 . The left ventricular tissues from the different groups were blinded and randomised to eliminate bias. A unique identification number was assigned to the left ventricular tissue of each animal and kept until all the measurements were completed. Briefly, left ventricular samples were defrosted in relaxing solution containing the following (mM): 100 KCl, 1.75 EGTA, 10 imidazole, 4 ATP-Mg, and 5 MgCl 2 ; pH 7.0. The left ventricular tissue was mechanically disrupted and incubated in relaxing solution supplemented with 1% Triton X-100 for 8 min to permeabilise the sarcolemmal membranes. The resulting isolated, skinned cardiomyocyte preparations were washed twice in relaxing solution. Thereafter, a skinned cardiomyocyte preparation (1-3 cells) was attached between a force transducer and a piezoelectric motor with silicon adhesive. Sarcomere length of the skinned cardiomyocyte preparation was adjusted to approximately 2.20 µm for isometric force measurements. The force was measured as a function of pCa (− log 10 [Ca 2+ ]) in solutions ranging from pCa 9.0 (relaxing solution) to pCa 4.5 (maximally activating solution).
Force was first measured in pCa 9.0 and 4.5 solutions and then in randomly ordered submaximal pCa solutions, followed by another measurement made in pCa 9.0 and 4.5 to assess any decline in cardiomyocyte performance. Measurements were continued in another three to four randomly ordered submaximal pCa solutions until a full force-pCa sigmoidal curve was obtained. This was followed by a last control measurement in pCa 9.0 and 4.5 to assess force rundown in the cardiomyocyte preparations. Skinned cardiomyocyte preparations without clear striations for sarcomere length measurement and that did not maintain more than 80% of maximal force at the end of the experimental protocol were discarded and data not used. For each activation, when steady-state force was reached, force was measured by abruptly slackened the skinned cardiomyocyte preparation by 20% of its initial length and transferred to pCa 9.0. Total force was calculated as the difference between steady-state force and the baseline force immediately after the skinned cardiomyocyte preparation was slacked. Active force www.nature.com/scientificreports/ was determined by subtracting passive force in pCa 9.0 from total force. Maximal force (force developed in pCa 4.5) and passive force (force developed in pCa 9.0) were normalised to the cross-sectional area (CSA) of the skinned cardiomyocyte preparation (CSA = 3.14 × (cell width/2) 2 ) . Active force at each pCa was expressed as a percentage of maximal force for that skinned cardiomyocyte preparation. Force-pCa relationships for each skinned cardiomyocyte preparation were fit to a modified Hill equation.
where F is steady state force, F max is the maximum isometric force at saturating Ca 2+ concentration, nH (Hill coefficient) is a measure of the steepness of the force-pCa relation which is an index of myofilament cooperative activation, pCa 50 is Ca 2+ concentration that produced 50% of maximal force which is used as an index of myofilament Ca 2+ sensitivity.
SERCA Ca 2+ uptake activity assay. Left ventricular tissue (~ 0.3 g) was mechanically disrupted in sarcoplasmic reticulum isolation buffer, which contained (mM): 290 sucrose, 10 imidazole, 3 NaN 3 , 0.5 PMSF, 0.5 DTT, 0.2 g/L trypsin inhibitor, and 0.2 g/L benzamide, pH 6.90. The tissue homogenate was centrifuged at 2300g for 15 min at 4 °C. The pellet was discarded and the supernatant was centrifuged at 50,000g for 3.5 h at 4 °C. After centrifugation, the supernatant was discarded and the pellet was re-suspended in 1 mL sarcoplasmic reticulum isolation buffer supplemented with 650 mM KCl, and incubated on ice for 45 min, followed by another centrifugation at 2300g for 10 min at 4 °C. The pellet was discarded and the supernatant was centrifuged at 50,000g for 4 h at 4 °C. The supernatant was discarded and the resulting pellet containing the isolated sarcoplasmic reticulum vesicles was re-suspended in isolation buffer, snap frozen in liquid nitrogen, and stored at − 80 °C for the SERCA Ca 2+ uptake assay. Ca 2+ uptake into the cardiac sarcoplasmic reticulum vesicles was measured according to the assay described by Beca et al. with some modifications 44 . The assay was performed at 30 °C in a 96-well plate with 100 µg of vesicles in a final volume of 100 µL of uptake buffer per well containing (mM): 100 KCl, 4 MgCl 2 , 20 HEPES, and 10 sodium oxalate, pH 7.0. The starting Ca 2+ concentration was adjusted to approximately 1.5 µM as assessed by the fluorescence ratio of fura-2. The reactions were initiated with a premix containing (mM): 1.5 Na 2 ATP, 1.5 creatine phosphate and 2 U/ml creatine phosphokinase. The fluorescence signals at 340 nm and 380 nm were recorded at 20-s intervals for 25 min with a multi-mode microplate reader (SpectraMax i3X, Molecular Devices). The rate of reaction for sarcoplasmic reticulum Ca 2+ uptake corrected for the amount of vesicle protein was calculated using the following formula: The data were plotted as rate of reaction against concentration of free Ca 2+ ([Ca 2+ ] free ), and the maximum Ca 2+ uptake rate was estimated from the plot by fitting a curve to the Michaelis-Menten equation.
The same uptake buffer was used for both diabetic and non-diabetic preparations, and the assay for both groups was run in parallel to minimise any potential variability in terms of the presence of heavy metals and Ca 2+ buffering capacity. In a separate experiment, thapsigargin, a potent SERCA inhibitor 45 completely inhibited Ca 2+ uptake in isolated sarcoplasmic reticulum preparation (see Supplementary Fig S2), confirming that the uptake of Ca 2+ was facilitated predominantly by SERCA. Sodium azide, a potent mitochondria inhibitor 46 did not affect Ca 2+ uptake, suggesting a lack of mitochondria-mediated Ca 2+ uptake in this assay (see Supplementary Fig S3).
Statistical analysis. Statistical analyses were performed using GraphPad Prism v.8.3.0. D' Agostino & Pearson test and Saphiro-Wilk test were used to check for a normal distribution. Student's unpaired t-tests were used to test for differences between non-diabetic and diabetic groups. Results are presented as means ± SEM, a value of P < 0.05 was considered statistically significant.