Diabetes Alters Intracellular Calcium Transients in Cardiac Endothelial Cells

Abdul Q. Sheikh; Jennifer R. Hurley; Wei Huang; Toloo Taghian; Andrei Kogan; Hongkwan Cho; Yigang Wang; Daria A. Narmoneva

doi:10.1371/journal.pone.0036840

Abstract

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca²⁺]_i) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca²⁺]_i homeostasis due to altered sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca²⁺ regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca²⁺]_i homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca²⁺]_i transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca²⁺ ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca²⁺]_i sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.

Citation: Sheikh AQ, Hurley JR, Huang W, Taghian T, Kogan A, Cho H, et al. (2012) Diabetes Alters Intracellular Calcium Transients in Cardiac Endothelial Cells. PLoS ONE 7(5): e36840. https://doi.org/10.1371/journal.pone.0036840

Editor: Gian Paolo Fadini, University of Padova, Italy

Received: November 10, 2011; Accepted: April 13, 2012; Published: May 9, 2012

Copyright: © 2012 Sheikh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NIDDK 1R21DK078814-01A1) (www.nih.gov); a Nanomedicine Fellowship, University of Cincinnati Nanomedicine Group (http://www.ceas.uc.edu/); an interdisciplinary faculty research grant, University of Cincinnati Research Council (http://webcentral.uc.edu/urc2/apply/programinfo.cfm); and startup funds from University of Cincinnati Biomedical Engineering (http://www.ceas.uc.edu/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Diabetes mellitus is a growing epidemic in the United States that afflicts 8.3% of the total US population [1]. Diabetic cardiomyopathy (DCM) is a diabetes-associated condition, which is defined as changes in the structure and function of the myocardium, independent of other etiological factors [2]. Despite the potential importance of DCM, the complex and multifactorial nature of the cellular and molecular perturbations, which may alter myocardial function, remain poorly understood.

Studies in animal models have shown that diabetes results in abnormal calcium (Ca²⁺) homeostasis that may precede clinically manifested DCM-related cardiac dysfunction [3], [4]. Importantly, alterations in Ca²⁺ regulatory mechanisms are a hallmark of cardiomyopathy and heart failure in human patients [4]. During normal myocardial contraction, intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiomyocytes increases due to Ca²⁺ entry via Ca²⁺ channels and inositol trisphosphate (IP3) mediated Ca²⁺ release from sarco/endoplasmic reticulum (SER). Cardiac relaxation is subsequently initiated by the Ca²⁺ decay process, which is governed by Ca²⁺ sequestration and Ca²⁺ efflux mechanisms. During Ca²⁺ sequestration, the sarcoplasmic reticulum Ca²⁺ ATPase pumps (SERCA) re-sequester the Ca²⁺ from the cytosol back to the SER. Similarly the Ca²⁺ efflux mechanism extrudes Ca²⁺ via plasma membrane Ca²⁺ ATPase pumps (PMCA), and sodium- calcium exchangers (NCX) [5]–[7].

In recent reports, diabetic cardiomyocytes displayed reduced SERCA activity and Ca²⁺ sequestration, as well as impaired NCX function [8], [9]. Similarly, aortic and arterial smooth muscle cells isolated from diabetic rats have shown reduced Ca²⁺ signaling [10] and abnormalities in SER Ca²⁺ mobilization [8]. Furthermore, SERCA overexpression in transgenic mice resulted in the protection against cardiac dysfunction in streptozotocin-induced diabetes [11]. Overall, these results suggest potential mechanisms for diabetes-induced disruptions in Ca²⁺ homeostasis.

It is well established that endothelium plays an essential role in vascular function, including nitric oxide (NO) -mediated modulation of vascular tone, control of adhesion between the vessel and cells circulating in the blood, and regulation of local cell and vessel growth via a variety of growth factors. Calcium is an important regulator of endothelial function that can mediate each of these pathways, including signaling via NO [12], [13], cell adhesion molecules [14], [15], and receptor tyrosine kinases [16], [17]. Importantly, diabetes is associated with impaired endothelium-dependent relaxation and diminished capacity to generate NO [18]–[21], elevated cell adhesion molecules [22], [23], and decreased angiogenic growth factors [24], [25]. Despite the available evidence of a link between calcium signaling and endothelial cell function, as well as documented endothelial dysfunction in diabetes, the possible effects of diabetes on Ca²⁺ signaling in endothelial cells are not known, which may delay the development of vascular therapies for diabetic patients. In particular, the information regarding the effects of diabetes on Ca²⁺ regulation in cardiac endothelium is especially important, because cardiac endothelial cells (CECs) are instrumental in normal myocardial function, including endocardium interactions, myocardial capillaries, vascular permeability and cardiac tissue remodeling. Therefore, this information is essential for a better understanding of mechanisms for the impaired cardiac function in DCM and for the development of new therapies to treat this condition.

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Figure 1. Effect of SERCA, NCX and PMCA inhibition on [Ca²⁺]_i transients in wild-type CECs.

[Ca²⁺]_i transients were evoked by UTP in the absence (A) or presence of SERCA (thapsigargin), NCX (benzamil) and PMCA (carboxyeosin (carbEO)) inhibitors in wild-type CECs (B, C, and D). Successful inhibition of SERCA, NCX and PMCA in wild-type CECs was demonstrated by an increased [Ca²⁺]_i decay time (E; from 80% to 20% of its peak amplitude) in the presence of inhibitors (Ca²⁺ free medium). [Ca²⁺]_i peak amplitude in the presence of NCX inhibitor was significantly greater then no inhibitor controls (F), while SERCA and PMCA inhibitors had no affect on peak amplitude (#: p<0.05; cell no.: A: 79, B: 88; C: 85, D: 92).

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Figure 2. Diabetes induces cardiac fibrosis and alters [Ca²⁺]_i transients in CECs.

(A): Analysis of the Picrosirius red staining of diabetic myocardium using polarized light microscopy showed the presence of cardiac fibrosis with significantly increased area of fibrillar collagen fibers (yellow, orange or red, middle panel), as compared to mostly reticular fibers (green, left panel) in the myocardium of age-matched wild-type animals (LV, RV: left or right ventricle). (B): Rats injected with STZ showed significant and rapid hyperglycemia by day 5 that persisted throughout the study. (C–F): Significant differences in UTP-evoked [Ca²⁺]_i transients were observed between wild-type and diabetic CECs (C, D). [Ca²⁺]_i transients in individual cells (outlined in white in the first frame) obtained using ratiometric analysis are shown as grey lines, with the average transient as a black line. Analysis of transients (E–F) demonstrated significantly longer [Ca²⁺]_i decay time and smaller peak amplitude in diabetic CECs, as compared to wild-type CECs (#: p<0.05; cell no: 206 (wt), 195 (db)).

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Figure 3. Diabetes alters SERCA-mediated Ca²⁺ sequestration.

Cells were treated with PMCA (carbEO) and NCX (benzamil) inhibitors to study SERCA activity during Ca²⁺ re-sequestration without the contribution of Ca²⁺ efflux mechanism. (A): Slow and long-lasting Ca²⁺ transients were evoked by thapsigargin (in Ca²⁺ free medium), with no significant difference in decay time between wild-type and diabetic CECs. However the peak amplitude in diabetic CEC was significantly smaller then in wild-type group. (B): UTP-evoked [Ca²⁺]_i transients in diabetic cells had significantly longer decay time and smaller peak amplitude in the absence or presence of extracellular Ca²⁺ (#: p<0.05; cell no: A: 82 (wt), 76 (db); B: 73 (wt), 78 (db)).

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Figure 4. Diabetes alters Ca²⁺ NCX activity without affecting PMCA-mediated efflux.

(A): CECs were treated with SERCA inhibitor (thapsigargin) to study the effect of diabetes on intracellular Ca²⁺ efflux mechanism without contributions from Ca²⁺ sequestration mechanism. UTP-evoked [Ca²⁺]_i decay time was significantly increased and the peak amplitude was decreased in diabetic CECs, as compared to wild-type controls. (B): CECs were treated with SERCA (thapsigargin) and NCX (benzamil) inhibitors to study the effect of diabetes on PMCA-mediated Ca²⁺ efflux. Under these conditions, there was no significant difference in UTP-evoked [Ca²⁺]_i transients between diabetic and wild-type CECs. (C): CECs were treated with SERCA (thapsigargin) and PMCA (carbEO) inhibitors to study the effect of diabetes on NCX-mediated Ca²⁺ efflux. In diabetic CECs, NCX activity was significantly reduced as indicated by increased decay time of UTP-evoked [Ca²⁺]_i transients. The peak amplitude was also significantly reduced in diabetic CECs under these conditions. (D): To confirm effect of diabetes on [Ca²⁺]_i transients, Ca²⁺ decay was inhibited by treating CECs with SERCA (thapsigargin), PMCA (carbEO) and NCX (benzamil) inhibitors. In wild-type CECs, [Ca²⁺]_i decay was completely blocked, while diabetic CECs still showed Ca²⁺ removal from the cytosol, with [Ca²⁺]_i decay time significantly faster than that in wild-type CECs. No change in peak amplitude was observed under these conditions (#: p<0.05; cell no. A: 69 (wt), 93 (db); B: 83 (wt), 73(db); C: 74 (wt), 71 (db); D 76 (wt), 72 (db)).

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Figure 5. Diabetes results in decreased NCX protein expression, with no difference in SERCA and PMCA expression levels.

Semi-quantitative analysis of immunofluorescent staining of CECs showed that diabetes significantly reduces the expression levels of NCX protein as compared to wild-type (NCX: red; blue: cell nuclei, #: p<0.05). No significant difference in the expression levels of SERCA and PMCA was observed in diabetic cells as compared to wild-type (SERCA: green; PMCA: yellow; blue: cell nuclei).

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The objective of this study was to determine the effect of diabetes on intracellular Ca²⁺ homeostasis in CECs in a rat model (streptozotocin-induced) of DCM. Uridine-5′-triphosphate (UTP) -evoked [Ca²⁺]_i transients were compared between diabetic and wild-type CECs under various combinations of SERCA, PMCA and NCX inhibitors. Our results demonstrate that diabetes results in altered Ca²⁺ homeostasis in endothelial cells via decreases in SERCA and NCX activities during Ca²⁺ sequestration and Ca²⁺ efflux, respectively.

Results

Effect of SERCA, NCX and PMCA inhibitors on [Ca²⁺]_i transients in wild-type CECs

The objective of these experiments was to establish the baseline for normal (wild-type) CEC response to SERCA, NCX and PMCA inhibitors. Consistent with previous studies [26], UTP (a P_2Y2 receptor agonist) evoked a [Ca²⁺]_i transient which decayed to basal level (Fig. 1A). The decay time of UTP-evoked [Ca²⁺]_i transient was significantly higher in the presence of SERCA inhibitor, thapsigargin (Fig. 1B, p<0.05). The effect of thapsigargin was further confirmed by the subsequent addition of 2.0 mmol/L Ca²⁺ to the medium, which evoked a marked [Ca²⁺]_i increase due to the depletion of SER and Ca²⁺ influx through store-operated Ca²⁺ channels (data not shown).

As expected, in the presence of NCX inhibitor (benzamil), the Ca²⁺ decay time (Fig. 1C) was significantly increased in CECs as compared to no inhibitor controls (Fig. 1E, p<0.05). Similarly, the presence of carboxyeosin (carbEO), a selective PMCA inhibitor resulted in a significant increase in the decay time of UTP-evoked [Ca²⁺]_i transient, as compared to no inhibitor treated CECs (Fig. 1D, E, p<0.05). These experiments were performed in Ca²⁺ free solution to avoid any contributions of Ca²⁺ influx from the extracellular space. To further validate these results, experiments were repeated in the presence of extracellular Ca²⁺, which also resulted in slower [Ca²⁺]_i decay time in wild-type CECs (data not shown) consistent with previous reports [27].

The [Ca²⁺]_i peak amplitude in wild-type CECs remained similar in the presence of SERCA and PMCA inhibitors as compared to no inhibitor controls. However, cells treated with NCX inhibitor showed a higher [Ca²⁺]_i peak as compared to no inhibitor controls (Fig. 1F, p<0.05).

Cardiac fibrosis and increase in blood glucose levels after STZ injection

Measurement of blood glucose levels after STZ injection showed significant and rapid hyperglycemia by day 5, which persisted until the end of the experiments (Fig. 2B). The blood glucose levels of the wild-type control animals remained in the physiological range for the duration of the study. Picrosirius red staining of the rat myocardium under polarized light showed significantly higher levels of cardiac fibrosis (yellow, orange, or red) in diabetic animals, as compared to tissue from age- and strain-matched wild-type control animals (p<0.05) [28], where mostly reticular, non-fibrillar collagen (green) was present (Fig. 2A). Interestingly, the values for the fibrillar collagen content in both groups are ∼2-fold higher than those reported in the literature [28], [29]. This may be due to the differences in methodology used to calculate collagen area fraction, including magnification (2X in this study vs. 200X in previous reports), the analysis of the sections of the whole heart vs. left ventricle only, and the normalization procedure (i.e. using the area obtained by tracing the myocardium to exclude voids in this study vs. the ratio of stained area to the unstained area in previous reports [28], [29]). Importantly, despite the differences in the absolute values, the relative increase (approximately 2.5-fold) in the fibrillar collagen content of the diabetic hearts as compared to the normal tissue is consistent with previous findings [28], [29].

[Ca²⁺]_i transients are disrupted in diabetic CECs

In wild-type CECs, UTP evoked a [Ca²⁺]_i transient, which gradually decayed with time (Fig. 2C, D). Interestingly, in diabetic CECs, the [Ca²⁺]_i transient decay was markedly delayed (Fig. 2C, D). Ratiometric analysis showed that in diabetic CECs, the decay time was significantly longer (Fig. 2E, p<0.05), and the [Ca²⁺]_i peak amplitude was significantly smaller, as compared to wild-type CECs (Fig. 2F, p<0.05).

Altered Ca²⁺ re-sequestration contributes to changes in [Ca²⁺]_i transients in diabetic CECs

[Ca²⁺]_i transients were first evoked by thapsigargin to explicitly examine Ca²⁺ depletion from SER in a Ca²⁺ free medium [30]. In diabetic cells, the peak amplitude was significantly smaller (db: 0.519±0.12 vs. wt: 0.723±0.05, cell no: 31 (db), 42(wt), p<0.05), and decay time was significantly longer (db: 131±15 vs. wt: 116±9, cell no: 42 (db), 38 (wt), p<0.05), as compared to wild-type CECs, indicating possible alterations in SERCA mediated Ca²⁺ responses in diabetic CECs.

To further examine SERCA-mediated Ca²⁺ sequestration without the contribution of Ca²⁺ efflux mechanism during [Ca²⁺]_i decay, CECs were treated with PMCA (carboxyeosin) and NCX (benzamil) inhibitors. Under these conditions, thapsigargin induced a slow and long-lasting Ca²⁺ transient in wild-type CECs. Diabetic CECs showed a similar response, with [Ca²⁺]_i decay time similar to that of wild-type CECs (Fig. 3A), while [Ca²⁺]_i peak amplitude in diabetic CECs was significantly smaller than in wild-type CECs (Fig. 3A, p<0.05). Interestingly, when [Ca²⁺]_i transient was evoked using UTP in the presence of PMCA (carboxyeosin) and NCX (benzamil) inhibitors, the [Ca²⁺]_i decay time in diabetic CECs was significantly increased and the peak amplitude was significantly decreased as compared to wild-type CECs (Fig. 3B, p<0.05). Results for [Ca²⁺]_i transients evoked in the presence of extracellular Ca²⁺ (2 mM CaCl₂ added) were similar, with significantly increased [Ca²⁺]_i decay time and decreased peak amplitude in diabetic CECs as compared to wild-type (Fig. 3B, p<0.05).

Ca²⁺ efflux mechanism is altered in diabetic CECs

CECs were treated with thapsigargin to block the contribution of the Ca²⁺ sequestration mechanism during [Ca²⁺]_i decay. The observed [Ca²⁺]_i decay time was significantly increased in diabetic CECs, as compared to wild-type cells (Fig. 4A, p<0.05), indicating impaired Ca²⁺ efflux in diabetic cells. A significant decrease in [Ca²⁺]_i peak amplitude was also observed in diabetic CECs (Fig. 4A, p<0.05). The subsequent experiments were performed to determine whether diabetes affects Ca²⁺ efflux mechanism either via altered PMCA or NCX activity, or a combination of both.

[Ca²⁺]_i extrusion by PMCA during Ca²⁺ efflux is not altered in diabetic CECs

To compare PMCA activity in wild-type and diabetic CECs, cells were treated with SERCA and NCX inhibitors (thapsigargin and benzamil, respectively) to exclude the contributions of SERCA-mediated Ca²⁺ sequestration and NCX-mediated Ca²⁺ efflux. Under these conditions, the UTP-evoked [Ca²⁺]_i transient did not show a significant difference in [Ca²⁺]_i decay time in diabetic CECs, as compared to wild-type (Fig. 4B, p<0.05), indicating that PMCA effectively extrudes Ca²⁺ in both diabetic and wild-type CECs. Furthermore, no differences in [Ca²⁺]_i peak amplitudes of diabetic and wild-type CECs were observed under these conditions.

Altered NCX activity affects Ca²⁺ efflux mechanism in diabetic CECs

[Ca²⁺]_i transients were evoked by UTP in the presence of SERCA and PMCA inhibitors (thapsigargin and carboxyeosin, respectively) to exclude contributions of SERCA-mediated Ca²⁺ sequestration and PMCA-mediated Ca²⁺ efflux. Interestingly, the [Ca²⁺]_i decay time was significantly increased in diabetic CECs as compared to wild-type (Fig. 4C, p<0.05), indicating altered NCX activity during the Ca²⁺ efflux. [Ca²⁺]_i peak amplitude in diabetic CECs was also significantly reduced as compared to wild-type CECs. To further confirm these results, [Ca²⁺]_i decay was inhibited by treating CECs with SERCA (thapsigargin), PMCA (carboxyeosin) and NCX (benzamil) inhibitors. Under these conditions, the [Ca²⁺]_i decay in wild-type CECs was completely blocked (Fig. 4D). However, in diabetic CECs, there was still Ca²⁺ removal from the cytosol, with [Ca²⁺]_i decay time significantly faster than in wild-type CECs (Fig. 4D, p<0.05). Under these conditions, no significant differences in the peak amplitudes of diabetic and wild-type CECs were observed. The reasons for the residual Ca²⁺ extrusion (Fig. 4D) are not clear, and further studies are required to investigate the possible cross talk between SERCA and PMCA pumps.

Diabetes alters NCX expression levels, but does not effect SERCA and NCX levels

Immunofluorescent staining for SERCA, NCX and PMCA protein expression levels indicated that NCX expression levels in diabetic CECs were significantly reduced as compared to wild-type CECs (Fig. 5, p<0.05), while no effects of diabetes on the expression levels of SERCA and PMCA proteins where observed (Fig. 5).

Discussion

The results of this study demonstrate that diabetes results in significantly altered [Ca²⁺]_i transients via disruptions in SERCA-mediated Ca²⁺ sequestration and NCX-mediated Ca²⁺ efflux mechanisms. To the best of our knowledge, this is the first report of the effect of diabetes on intracellular Ca²⁺ regulatory mechanisms and overall Ca²⁺ homeostasis in the cardiac endothelium. These results are consistent with previous studies in diabetic cardiomyocytes, where altered Ca²⁺ handling was observed in cells isolated from diabetic mice [31] and rats [32]. Similarly, smooth muscle cells isolated from the aortas of type I diabetic rats showed significant alterations in SER-Ca²⁺ kinetics, resulting in the development of diabetic vascular complications [8]. Our results demonstrate that in diabetic CECs, alterations in [Ca²⁺]_i transients involve a change in the Ca²⁺ sequestration mechanism. This change in Ca²⁺ sequestration is due to the reduced activity of SERCA to re-sequester Ca²⁺ upon UTP activation. The effect of reduced SERCA activity during Ca²⁺ sequestration in diabetic CECs appeared more prominent during Ca²⁺ decay, which may be due to the fact that SERCA has a higher affinity for Ca²⁺ as compared to PMCA [5], [7], [33]. Our results suggest that the reduced Ca²⁺ sequestration was independent of SERCA protein expression, since staining for SERCA protein remained unchanged in diabetic CECs. Taken together, these results indicate a common mechanism for diabetes-induced alterations in Ca²⁺ sequestration among several cell types, including cardiomyocytes, endothelial cells and smooth muscle cells.

Our results show that NCX activity during Ca²⁺ efflux is altered in diabetic CECs, as compared to wild-type controls. The exact role of the NCX in endothelial cells is still unclear. However, it is generally accepted that this exchanger normally functions (during the resting stage or when [Ca²⁺]_i is high) to extrude Ca²⁺ out of the cell [33]. Because of its bidirectional property, it is also thought to play a critical role in Ca²⁺ influx when the intracellular Na⁺ concentration is high enough to drive the NCX. Our findings of a decrease in NCX activity (with no changes in PMCA activity) in diabetic CECs suggest that the ability of diabetic CECs to maintain Ca²⁺ homeostasis is compromised, which may contribute to abnormal cardiac function. This reduced ability of NCX to extrude Ca²⁺ may also be related to the reduced NCX protein levels as shown by the immunofluorescent staining analysis of diabetic CECs. Our results of altered NCX activity and reduced protein expression levels in diabetic CECs are consistent with previous studies in cardiomyocytes [34], [35]. In contrast, Hill et al. [36] showed that in porcine coronary artery smooth muscle cells, the diabetic condition does not affect Ca²⁺ efflux via NCX, while resulting in altered Ca²⁺ efflux via PMCA. Our results also suggest that in diabetic CECs, PMCA effectively extrudes Ca²⁺ in the presence of SERCA and NCX inhibitors.

Our results show that while diabetic conditions result in impaired function of SERCA and NCX, there are no differences in PMCA function and protein expression between diabetic and wild-type cells. These results are consistent with those by Hill et al. [36], where PMCA activity in diabetic smooth muscle cells was similar to that in wild-type cells in the absence of a functional SERCA. Our results also corroborate those of van Breemen et al. [37] in smooth muscle cells, which demonstrated that upon inhibition of NCX and SERCA, Ca²⁺ is primarily extruded by the PMCA. Overall, our findings suggest that abnormal endothelium-dependent relaxation and diminished capacity to generate NO in diabetes [19] may result from altered Ca²⁺ transients due to impaired NCX and SERCA, which can reduce activation of eNOS and NO production by endothelial cells [38]. Also, altered Ca²⁺ transients in diabetic cells may affect ICAM-1-coupled cytoskeletal rearrangements and endothelial cell interactions with inflammatory cells [14], consistent with upregulation of ICAM-1 observed in diabetic patients [22].

In this study, we specifically focused on the effects of DCM on intracellular calcium regulatory mechanisms (calcium sequestration and efflux), and therefore, the experiments were performed in the absence of extracellular calcium (unless otherwise stated). However, in order to elucidate the mechanisms for DCM-induced alterations in overall calcium signaling, information regarding the effects of diabetic conditions on store operated calcium entry is important as well, and will be the subject of future studies.

While the development of DCM and the associated mechanisms are complex and multifactorial, impaired Ca²⁺ homeostasis has been identified as a hallmark abnormality for this disorder [3]. Numerous approaches have been suggested to treat DCM by increasing SERCA and NCX expression [11]. In addition, Ca²⁺ dependent signaling pathways (such as PKC activity [39]) have been identified as possible therapeutic strategies. Information regarding how diabetes affects Ca²⁺ homeostasis in cardiac endothelium is paramount because Ca²⁺ dependent pathways in CECs are involved in endocardium interactions, vascularization and nitric oxide-mediated cardiac hypertrophy. Furthermore, it has been recently shown that endothelial cells may be directly involved in diabetes–induced cardiac fibrosis via endothelial-to-mesenchymal transition [40]. Therefore, the results of this study provide a new insight into the effects of diabetes on Ca²⁺ regulation in CECs, which may contribute to the design of therapeutic approaches to target abnormal Ca²⁺ homeostasis in diabetic cardiomyopathy. Further studies will be required to determine the exact molecular mechanisms for the observed effects.

Materials and Methods

Ethics statement

All procedures were performed according to the protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee (IACUC Protocol # 09-06-24-01).

Animal model of type I diabetes

In this study, a rat model of type I (streptozotocin-induced) diabetes and non-atherogenic diabetic cardiomyopathy [41] was used. Eight-week-old female Sprague Dawley rats (SAS SD Strain 400, Charles River) were injected intraperitoneally with 70 mg/kg streptozotocin (STZ, Sigma-Aldrich) and allowed to develop type I diabetes for six weeks [41], with diabetic phenotype confirmed by serum glucose levels greater than 300 mg/dl. To assess DCM-associated cardiac fibrosis, fibrillar collagen content was measured using picrosirius red staining of paraffin sections of myocardium [42]. The stained sections were observed under polarized light and photographed with the same exposure time for each section. The percentage of fibrillar collagen (yellow, red or orange color) or non-fibrillar collagen (green color) relative to the total area of the myocardium (traced by hand to exclude void areas) was determined using computer-assisted analysis (Adobe Photoshop CS3, Adobe Systems Incorporated, CA).

Cardiac endothelial cell isolation and culture

Cardiac cells were isolated from the myocardium of diabetic and wild-type rats as described previously [43]. CECs were sorted from cardiac cell mixture using PECAM-1- and ICAM-2-conjugated magnetic beads (Invitrogen Corporation, CA). Endothelial phenotype was confirmed using von Willebrand Factor staining (Sigma-Aldrich, MO), with >95% cells positively stained. CECs were cultured in medium M199 (HyClone, UT) with 10% fetal bovine serum (Atlanta Biologicals, GA), 1% antibiotic/antimycotic (AtlantaBiologicals, GA), 1% heparin (Sigma-Aldrich, MO) and 10 ng/mL endothelial growth factor supplement (Sigma-Aldrich, MO). CECs from passages 4–9 were used for all experiments. Prior to Ca²⁺ experiments, CECs were trypsinized and seeded on multi-well glass coverslips (Sigma-Aldrich, MO) for 24 hours.

Measurements of [Ca²⁺]_i transients

To exclude the contribution of extracellular Ca²⁺ influx during [Ca²⁺]_i transient, experiments were performed in a Ca²⁺ free medium (Hanks’ balanced salt solution without Ca²⁺ and Mg²⁺, supplemented with 25 mM HEPES (Invitrogen Corporation, CA), 0.5% bovine serum albumin (Atlanta Biologicals, GA), 2.5 mM probenecid (Sigma-Aldrich, MO), pH 7.45) and 0.5 mM EGTA (calcium buffer), unless otherwise stated. Wild-type and diabetic CECs were loaded with 5 μM Ca²⁺ sensitive indicator Fura-2 AM (Sigma-Aldrich, MO) for 40 minutes at room temperature. In some experiments, extracellular Ca²⁺ was incorporated by adding 2 mM CaCl₂ in the Ca²⁺ free medium without EGTA. [Ca²⁺]_i transients were evoked in CECs by adding 10 µM UTP (Invitrogen Corporation, CA). UTP has been shown to act on P-type purinergic receptors (P_2Y2), which leads to a G protein-mediated activation of phospholipase C, followed by inositol 1,4,5-trisphosphate (IP3) formation and the release of Ca²⁺ from IP3 sensitive Ca²⁺ stores (mainly from SER) [26]. The imaging setup included an Olympus IX81 inverted fluorescent microscope (Olympus, PA) equipped with cooled charged couple device camera, automated filter wheel (340 nm and 380 nm excitation wavelengths) and a Fura-2 filter cube (400 diachronic and 510 nm wide band emission filter). After UTP administration, images at 340 nm (bound Ca2+) and 380 nm (unbound Ca²⁺) excitation wavelengths were recorded at 510 nm emission after every 250 msec for at least 20 minutes. For each time point, F340/F380 ratiometric analysis was performed by determining the ratio of 340 nm and the corresponding 380 nm image (ImagePro software, Media Cybernetics Inc., MD). Regions of interest outlining cell boundaries on the ratio image were defined, and the variations in fluorescent intensity of selected cells were tabulated [44]. The individual traces (shown in gray, Fig. 2D) were then used to obtain the average intensity as a function of time (shown in black in Fig. 2D). For every treatment group, each experiment was repeated at least 3 times using fresh cells, and at least n = 70 individual cell traces were analyzed to obtain the average intensity plot as shown in Fig. 2D. Standard deviation was calculated at every time point, and the representative SD error bars are shown for every 10^th point.

Inhibitor studies

To assess the activities of SERCA, PMCA and NCX during the decay of [Ca²⁺]_i transients, wild-type CECs were treated with 1 μM thapsigargin (Sigma-Aldrich, MO), a selective SERCA inhibitor [45], 100 μM benzamil (Sigma-Aldrich, MO), an NCX inhibitor [46] or 25 μM carboxyeosin ester (carbEO, Sigma-Aldrich, MO), a selective PMCA inhibitor [47] (Fig. 1). Cells were incubated in an inhibitor-containing medium for 30 minutes prior to Fura-2 administration. For Ca²⁺ sequestration mechanism studies (SERCA activity), [Ca²⁺]_i transients were first evoked using 1 μM thapsigargin, followed by 10 μM UTP in a Ca²⁺ free medium. During these experiments, the Ca²⁺ efflux was inhibited using PMCA and NCX inhibitors at all times. To examine the Ca²⁺ efflux mechanism (PMCA and NCX activities), diabetic and wild-type CECs were treated with NCX inhibitor (benzamil) or PMCA inhibitor (carboxyeosin). These experiments were performed under the following conditions: a) SERCA activity was inhibited at all times (thapsigargin present in the medium), b) NCX activity was inhibited to examine PMCA activity, and c) PMCA activity was inhibited to examine NCX activity. In these experiments, UTP was used to evoke [Ca²⁺]_i transients.

Immunofluorescent staining and semi-quantitative analysis

To investigate the effect of diabetes on the protein expression levels of SERCA, NCX and PMCA, diabetic and wild-type CECs were seeded in 24 well plates (6 wells per cell type) and allowed to spread. At 24 hours after seeding, cells were fixed in 2% paraformaldehyde. Immunofluorescent staining was performed by incubating the cells in rabbit anti-rat antibodies against SERCA, NCX and PMCA (abcam, MA) proteins, followed by incubation with Alexa Fluor 568-conjugated anti-rabbit secondary antibody (Invitrogen Corporation, CA). Cells were also stained with DAPI to identify cell nuclei (Invitrogen Corporation, CA). A total of 20 images for each protein were captured at 20X magnification using an Olympus IX81 microscope. The average fluorescent intensity of each image was quantified using MATLAB software (The Math Works, MA), and normalized by the intensity value of the corresponding negative control image (CECs incubated with only secondary antibody).

Statistical analyses

The decay time of agonist-evoked Ca²⁺ transient was determined as the time in which [Ca²⁺]_i decayed from 80% to 20% of its peak amplitude (presented as t_80–20). The results are expressed as mean ± SD. One way ANOVA (SPSS, IBM, IL) was used to determine the effect of diabetes on [Ca²⁺]_i decay kinetics and protein expression levels in CECs. A p<0.05 was considered to be statistically significant. All experiments were repeated at least 3 times; at least 70 diabetic/wild-type CECs were analyzed per treatment.

Author Contributions

Conceived and designed the experiments: AQS DAN. Performed the experiments: AQS JRH WH TT. Analyzed the data: AQS HC DAN. Contributed reagents/materials/analysis tools: AQS WH YW AK DAN. Wrote the paper: AQS DAN. Contributed to discussion and manuscript proofreading: AK. Instrumental in performing animal studies and maintaining cell bank: JRH. Contributed to revised manuscript preparation and proofreading: JRH.

References

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- Google Scholar
27. Moccia F, Berra-Romani R, Baruffi S, Spaggiari S, Signorelli S, et al. (2002) Ca2+ uptake by the endoplasmic reticulum Ca2+-ATPase in rat microvascular endothelial cells. Biochemical Journal 364: 235–244.
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- Google Scholar
28. van Linthout S, Seeland U, Riad A, Eckhardt O, Hohl M, et al. (2008) Reduced MMP-2 activity contributes to cardiac fibrosis in experimental diabetic cardiomyopathy. Basic Research in Cardiology 103: 319–327.
- View Article
- Google Scholar
29. Westermann D, Van Linthout S, Dhayat S, Dhayat N, Escher F, et al. (2007) Cardioprotective and anti-inflammatory effects of interleukin converting enzyme inhibition in experimental diabetic cardiomyopathy. Diabetes 56: 1834–1841.
- View Article
- Google Scholar
30. Gonzalez De La Fuente P, Savineau JP, Marthan R (1995) Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum Ca2+ pump blockers: Thapsigargin and cyclopiazonic acid. Pflugers Archiv European Journal of Physiology 429: 617–624.
- View Article
- Google Scholar
31. Pereira L, Matthes J, Schuster I, Valdivia HH, Herzig S, et al. (2006) Mechanisms of [Ca2+]i transient decrease in cardiomyopathy of db/db type 2 diabetic mice. Diabetes 55: 608–615.
- View Article
- Google Scholar
32. Zhao XY, Hu SJ, Li J, Mou Y, Chen BP, et al. (2006) Decreased cardiac sarcoplasmic reticulum Ca2+-ATPase activity contributes to cardiac dysfunction in streptozotocin-induced diabetic rats. Journal of Physiology and Biochemistry 62: 1–8.
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- Google Scholar
34. Chattou S, Diacono J, Feuvray D (1999) Decrease in sodium-calcium exchange and calcium currents in diabetic rat ventricular myocytes. Acta Physiologica Scandinavica 166: 137–144.
- View Article
- Google Scholar
35. Choi KM, Zhong Y, Hoit BD, Grupp IL, Hahn H, et al. (2002) Defective intracellular Ca2+ signaling contributes to cardiomyopathy in type 1 diabetic rats. American Journal of Physiology – Heart and Circulatory Physiology 283: H1398.
- View Article
- Google Scholar
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- Google Scholar
37. Van Breemen C (1989) Cellular mechanisms regulating [Ca2+](i) smooth muscle. Annual Review of Physiology 51: 315–329.
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- Google Scholar
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- Google Scholar
39. Dang L, Seale JP, Qu X (2005) High glucose-induced human umbilical vein endothelial cell hyperpermeability is dependent on protein kinase C activation and independent of the Ca2+-nitric oxide signalling pathway. Clinical and Experimental Pharmacology and Physiology 32: 771–776.
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- Google Scholar
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- View Article
- Google Scholar
41. Penpargkul S, Schaible T, Yipintsoi T, Scheuer J (1980) The effect of diabetes on performance and metabolism of rat hearts. Circulation Research 47: 911–921.
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42. Xavier-Vidal R (2005) Oxytalan elastic and collagen fibers during the repair process in experimental nitric oxide inhibition. Clinics (São Paulo, Brazil) 60: 85–92.
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- Google Scholar
43. Nishida M, Carley WW, Gerritsen ME, Ellingsen O, Kelly RA, et al. (1993) Isolation and characterization of human and rat cardiac microvascular endothelial cells. American Journal of Physiology – Heart and Circulatory Physiology 264: H639–H652.
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- Google Scholar
44. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260: 3440–3450.
- View Article
- Google Scholar
45. Yamamoto N, Watanabe H, Kakizawa H, Hirano M, Kobayashi A, et al. (1995) A study on thapsigargin-induced calcium ion and cation influx pathways in vascular endothelial cells. Biochimica et Biophysica Acta – Molecular Cell Research 1266: 157–162.
- View Article
- Google Scholar
46. Siegl PKS, Cragoe EJ Jr, Trumble MJ, Kaczorowski GJ (1984) Inhibition of Na+/Ca2+ exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride. Proceedings of the National Academy of Sciences of the United States of America 81: 3238–3242.
- View Article
- Google Scholar
47. Green AK, Cobbold PH, Dixon CJ (1997) Effects on the hepatocyte [Ca2+](i) oscillator of inhibition of the plasma membrane Ca2+ pump by carboxyeosin or glucagon-(19–29). Cell Calcium 22: 99–109.
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[ref1] 1. Association AD (2011) ADA fact sheet.

[ref2] 2. Aneja A, Tang WHW, Bansilal S, Garcia MJ, Farkouh ME (2008) Diabetic Cardiomyopathy: Insights into Pathogenesis, Diagnostic Challenges, and Therapeutic Options. American Journal of Medicine 121: 748.
View Article
Google Scholar

[3] View Article

[4] Google Scholar

[ref3] 3. Lebeche D, Davidoff AJ, Hajjar RJ (2008) Interplay between impaired calcium regulation and insulin signaling abnormalities in diabetic cardiomyopathy. Nature Clinical Practice Cardiovascular Medicine 5: 715–724.
View Article
Google Scholar

[6] View Article

[7] Google Scholar

[ref4] 4. Morgan JP, Erny RE, Allen PD, Grossman W, Gwathmey JK (1990) Abnormal intracellular calcium handling, a major cause of systolic and diastolic dysfunction in ventricular myocardium from patients with heart failure. Circulation 81: 21–32.
View Article
Google Scholar

[9] View Article

[10] Google Scholar

[ref5] 5. Inesi G, Prasad AM, Pilankatta R (2008) The Ca2+ ATPase of cardiac sarcoplasmic reticulum: Physiological role and relevance to diseases. Biochemical and Biophysical Research Communications 369: 182–187.
View Article
Google Scholar

[12] View Article

[13] Google Scholar

[ref6] 6. Brini M, Carafoli E (2011) The plasma membrane Ca2+ ATPase and the plasma membrane sodium calcium exchanger cooperate in the regulation of cell calcium. Cold Spring Harbor perspectives in biology 3:
View Article
Google Scholar

[15] View Article

[16] Google Scholar

[ref7] 7. Di Leva F, Domi T, Fedrizzi L, Lim D, Carafoli E (2008) The plasma membrane Ca2+ ATPase of animal cells: Structure, function and regulation. Archives of Biochemistry and Biophysics 476: 65–74.
View Article
Google Scholar

[18] View Article

[19] Google Scholar

[ref8] 8. Ma L, Zhu B, Chen X, Liu J, Guan Y, et al. (2008) Abnormalities of sarcoplasmic reticulum CA2+ mobilization in aortic smooth muscle cells from streptozotocin-induced diabetic rats. Clinical and Experimental Pharmacology and Physiology 35: 568–573.
View Article
Google Scholar

[21] View Article

[22] Google Scholar

[ref9] 9. Hattori Y, Matsuda N, Kimura J, Ishitani T, Tamada A, et al. (2000) Diminished function and expression of the cardiac Na+-Ca2+ exchanger in diabetic rats: Implication in Ca2+ overload. Journal of Physiology 527: 85–94.
View Article
Google Scholar

[24] View Article

[25] Google Scholar

[ref10] 10. Searls YM, Loganathan R, Smirnova IV, Stehno-Bittel L (2010) Intracellular Ca2+ regulating proteins in vascular smooth muscle cells are altered with type 1 diabetes due to the direct effects of hyperglycemia. Cardiovascular Diabetology 9:
View Article
Google Scholar

[27] View Article

[28] Google Scholar

[ref11] 11. Trost SU, Belke DD, Bluhm WF, Meyer M, Swanson E, et al. (2002) Overexpression of the sarcoplasmic reticulum CA2+-ATPase improves myocardial contractility in diabetic cardiomyopathy. Diabetes 51: 1166–1171.
View Article
Google Scholar

[30] View Article

[31] Google Scholar

[ref12] 12. Fleming I, Busse R (2003) Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. American Journal of Physiology – Regulatory Integrative and Comparative Physiology 284: R1–R12.
View Article
Google Scholar

[33] View Article

[34] Google Scholar

[ref13] 13. Lin S, Fagan KA, Li KX, Shaul PW, Cooper DMF, et al. (2000) Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry. Journal of Biological Chemistry 275: 17979–17985.
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref14] 14. Etienne-Manneville S, Manneville JB, Adamson P, Wilbourn B, Greenwood J, et al. (2000) ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines. Journal of Immunology 165: 3375–3383.
View Article
Google Scholar

[39] View Article

[40] Google Scholar

[ref15] 15. Cook-Mills JM, Johnson JD, Deem TL, Ochi A, Wang L, et al. (2004) Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity. Biochemical Journal 378: 539–547.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref16] 16. Improta-Brears T, Whorton AR, Codazzi F, York JD, Meyer T, et al. (1999) Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium. Proceedings of the National Academy of Sciences of the United States of America 96: 4686–4691.
View Article
Google Scholar

[45] View Article

[46] Google Scholar

[ref17] 17. Fleming I, Busse R (1997) Tyrosine phosphorylation and bradykinin induced signaling in endothelial cells. American Journal of Cardiology 80: 102A–109A.
View Article
Google Scholar

[48] View Article

[49] Google Scholar

[ref18] 18. Cosentino F, Lüscher TF (1998) Endothelial dysfunction in diabetes mellitus. Journal of Cardiovascular Pharmacology 32: S54–S61.
View Article
Google Scholar

[51] View Article

[52] Google Scholar

[ref19] 19. Huszka M, Kaplar M, Rejto L, Tornai I, Palatka K, et al. (1997) The association of reduced endothelium derived relaxing factor-no production with endothelial damage and increased in vivo platelet activation in patients with diabetes mellitus. Thrombosis Research 86: 173–180.
View Article
Google Scholar

[54] View Article

[55] Google Scholar

[ref20] 20. Obrosova IG, Drel VR, Oltman CL, Mashtalir N, Tibrewala J, et al. (2007) Role of nitrosative stress in early neuropathy and vascular dysfunction in streptozotocin-diabetic rats. American Journal of Physiology – Endocrinology and Metabolism 293: E1645–E1655.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref21] 21. Goel A, Zhang Y, Anderson L, Rahimian R (2007) Gender difference in rat aorta vasodilation after acute exposure to high glucose: Involvement of protein kinase C β and superoxide but not of Rho Kinase. Cardiovascular Research 76: 351–360.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref22] 22. El Amine M, Sohawon S, Lagneau L, Gaham N, Berkenboom G, et al. (2010) Plasma levels of icam-1 and circulating endothelial cells are elevated in unstable types 1 and 2 diabetes. Endocrine Regulations 44: 17–24.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref23] 23. Gustavsson C, Agardh CD, Zetterqvist AV, Nilsson J, Agardh E, et al. (2010) Vascular cellular adhesion molecule-1 (VCAM-1) expression in mice retinal vessels is affected by both hyperglycemia and hyperlipidemia. PLoS ONE 5: 1–12.
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref24] 24. Arnalich F, Maldifassi MC, Atienza G, Ciria E, Quesada A, et al. (2011) Decreased vascular endothelial growth factor response to acute hypoglycemia in type 2 diabetic patients with hypoglycemic coma. Cytokine.

[ref25] 25. Tchaikovski V, Olieslagers S, Böhmer FD, Waltenberger J (2009) Diabetes mellitus activates signal transduction pathways resulting in vascular endothelial growth factor resistance of human monocytes. Circulation 120: 150–159.
View Article
Google Scholar

[70] View Article

[71] Google Scholar

[ref26] 26. Moccia F, Baruffi S, Spaggiari S, Coltrini D, Berra-Romani R, et al. (2001) P2Y1 and P2Y2 receptor-operated Ca2+ signals in primary cultures of cardiac microvascular endothelial cells. Microvascular Research 61: 240–252.
View Article
Google Scholar

[73] View Article

[74] Google Scholar

[ref27] 27. Moccia F, Berra-Romani R, Baruffi S, Spaggiari S, Signorelli S, et al. (2002) Ca2+ uptake by the endoplasmic reticulum Ca2+-ATPase in rat microvascular endothelial cells. Biochemical Journal 364: 235–244.
View Article
Google Scholar

[76] View Article

[77] Google Scholar

[ref28] 28. van Linthout S, Seeland U, Riad A, Eckhardt O, Hohl M, et al. (2008) Reduced MMP-2 activity contributes to cardiac fibrosis in experimental diabetic cardiomyopathy. Basic Research in Cardiology 103: 319–327.
View Article
Google Scholar

[79] View Article

[80] Google Scholar

[ref29] 29. Westermann D, Van Linthout S, Dhayat S, Dhayat N, Escher F, et al. (2007) Cardioprotective and anti-inflammatory effects of interleukin converting enzyme inhibition in experimental diabetic cardiomyopathy. Diabetes 56: 1834–1841.
View Article
Google Scholar

[82] View Article

[83] Google Scholar

[ref30] 30. Gonzalez De La Fuente P, Savineau JP, Marthan R (1995) Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum Ca2+ pump blockers: Thapsigargin and cyclopiazonic acid. Pflugers Archiv European Journal of Physiology 429: 617–624.
View Article
Google Scholar

[85] View Article

[86] Google Scholar

[ref31] 31. Pereira L, Matthes J, Schuster I, Valdivia HH, Herzig S, et al. (2006) Mechanisms of [Ca2+]i transient decrease in cardiomyopathy of db/db type 2 diabetic mice. Diabetes 55: 608–615.
View Article
Google Scholar

[88] View Article

[89] Google Scholar

[ref32] 32. Zhao XY, Hu SJ, Li J, Mou Y, Chen BP, et al. (2006) Decreased cardiac sarcoplasmic reticulum Ca2+-ATPase activity contributes to cardiac dysfunction in streptozotocin-induced diabetic rats. Journal of Physiology and Biochemistry 62: 1–8.
View Article
Google Scholar

[91] View Article

[92] Google Scholar

[ref33] 33. Blaustein MP, Lederer WJ (1999) Sodium/calcium exchange: Its physiological implications. Physiological Reviews 79: 763–854.
View Article
Google Scholar

[94] View Article

[95] Google Scholar

[ref34] 34. Chattou S, Diacono J, Feuvray D (1999) Decrease in sodium-calcium exchange and calcium currents in diabetic rat ventricular myocytes. Acta Physiologica Scandinavica 166: 137–144.
View Article
Google Scholar

[97] View Article

[98] Google Scholar

[ref35] 35. Choi KM, Zhong Y, Hoit BD, Grupp IL, Hahn H, et al. (2002) Defective intracellular Ca2+ signaling contributes to cardiomyopathy in type 1 diabetic rats. American Journal of Physiology – Heart and Circulatory Physiology 283: H1398.
View Article
Google Scholar

[100] View Article

[101] Google Scholar

[ref36] 36. Hill BJF, Price EM, Dixon JL, Sturek M (2003) Increased calcium buffering in coronary smooth muscle cells from diabetic dyslipidemic pigs. Atherosclerosis 167: 15–23.
View Article
Google Scholar

[103] View Article

[104] Google Scholar

[ref37] 37. Van Breemen C (1989) Cellular mechanisms regulating [Ca2+](i) smooth muscle. Annual Review of Physiology 51: 315–329.
View Article
Google Scholar

[106] View Article

[107] Google Scholar

[ref38] 38. Bossu JL, Elhamdani A, Feltz A (1992) Voltage-dependent calcium entry in confluent bovine capillary endothelial cells. FEBS Letters 299: 239.
View Article
Google Scholar

[109] View Article

[110] Google Scholar

[ref39] 39. Dang L, Seale JP, Qu X (2005) High glucose-induced human umbilical vein endothelial cell hyperpermeability is dependent on protein kinase C activation and independent of the Ca2+-nitric oxide signalling pathway. Clinical and Experimental Pharmacology and Physiology 32: 771–776.
View Article
Google Scholar

[112] View Article

[113] Google Scholar

[ref40] 40. Widyantoro B, Emoto N, Nakayama K, Anggrahini DW, Adiarto S, et al. (2010) Endothelial cell-derived endothelin-1 promotes cardiac fibrosis in diabetic hearts through stimulation of endothelial-to-mesenchymal transition. Circulation 121: 2407–2418.
View Article
Google Scholar

[115] View Article

[116] Google Scholar

[ref41] 41. Penpargkul S, Schaible T, Yipintsoi T, Scheuer J (1980) The effect of diabetes on performance and metabolism of rat hearts. Circulation Research 47: 911–921.
View Article
Google Scholar

[118] View Article

[119] Google Scholar

[ref42] 42. Xavier-Vidal R (2005) Oxytalan elastic and collagen fibers during the repair process in experimental nitric oxide inhibition. Clinics (São Paulo, Brazil) 60: 85–92.
View Article
Google Scholar

[121] View Article

[122] Google Scholar

[ref43] 43. Nishida M, Carley WW, Gerritsen ME, Ellingsen O, Kelly RA, et al. (1993) Isolation and characterization of human and rat cardiac microvascular endothelial cells. American Journal of Physiology – Heart and Circulatory Physiology 264: H639–H652.
View Article
Google Scholar

[124] View Article

[125] Google Scholar

[ref44] 44. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260: 3440–3450.
View Article
Google Scholar

[127] View Article

[128] Google Scholar

[ref45] 45. Yamamoto N, Watanabe H, Kakizawa H, Hirano M, Kobayashi A, et al. (1995) A study on thapsigargin-induced calcium ion and cation influx pathways in vascular endothelial cells. Biochimica et Biophysica Acta – Molecular Cell Research 1266: 157–162.
View Article
Google Scholar

[130] View Article

[131] Google Scholar

[ref46] 46. Siegl PKS, Cragoe EJ Jr, Trumble MJ, Kaczorowski GJ (1984) Inhibition of Na+/Ca2+ exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride. Proceedings of the National Academy of Sciences of the United States of America 81: 3238–3242.
View Article
Google Scholar

[133] View Article

[134] Google Scholar

[ref47] 47. Green AK, Cobbold PH, Dixon CJ (1997) Effects on the hepatocyte [Ca2+](i) oscillator of inhibition of the plasma membrane Ca2+ pump by carboxyeosin or glucagon-(19–29). Cell Calcium 22: 99–109.
View Article
Google Scholar

[136] View Article

[137] Google Scholar

Figures

Abstract

Introduction

Results

Effect of SERCA, NCX and PMCA inhibitors on [Ca2+]i transients in wild-type CECs

Cardiac fibrosis and increase in blood glucose levels after STZ injection

[Ca2+]i transients are disrupted in diabetic CECs

Altered Ca2+ re-sequestration contributes to changes in [Ca2+]i transients in diabetic CECs

Ca2+ efflux mechanism is altered in diabetic CECs

[Ca2+]i extrusion by PMCA during Ca2+ efflux is not altered in diabetic CECs

Altered NCX activity affects Ca2+ efflux mechanism in diabetic CECs

Diabetes alters NCX expression levels, but does not effect SERCA and NCX levels

Discussion

Materials and Methods

Ethics statement

Animal model of type I diabetes

Cardiac endothelial cell isolation and culture

Measurements of [Ca2+]i transients

Inhibitor studies

Immunofluorescent staining and semi-quantitative analysis

Statistical analyses

Author Contributions

References

Effect of SERCA, NCX and PMCA inhibitors on [Ca²⁺]_i transients in wild-type CECs

[Ca²⁺]_i transients are disrupted in diabetic CECs

Altered Ca²⁺ re-sequestration contributes to changes in [Ca²⁺]_i transients in diabetic CECs

Ca²⁺ efflux mechanism is altered in diabetic CECs

[Ca²⁺]_i extrusion by PMCA during Ca²⁺ efflux is not altered in diabetic CECs

Altered NCX activity affects Ca²⁺ efflux mechanism in diabetic CECs

Measurements of [Ca²⁺]_i transients