Mitochondrial Ca 2+ uptake affects contractile properties in streptozotocin-induced diabetic rat myocardium

Mitochondrial Ca 2+ uptake affects contractile properties in streptozotocin-induced diabetic rat myocardium | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mitochondrial Ca 2+ uptake affects contractile properties in streptozotocin-induced diabetic rat myocardium Haruka Sato, Sana Koyama, Ayana Matsumoto, Shiori Akazawa, Kao Takeda, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9025606/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Diabetes mellitus is a major risk factor for the development of heart failure. However, the role of mitochondrial Ca 2+ in regulating contractile function in diabetic myocardium has not been fully elucidated. In the present study, we examined how mitochondrial Ca 2+ uptake influences myocardial contractile properties under diabetic conditions. Methods and Results: Rats were injected with 55 mg/kg streptozotocin (STZ rats) or solvent (Ctr rats). Six weeks after the injection, trabeculae were dissected from the right ventricles. Force was recorded with a strain gauge, intracellular Ca 2+ with fura-2, reactive oxygen species (ROS) production with MitoSOX Red, and mitochondrial Ca 2+ with rhod-2 in trabeculae. The maximal velocity of contraction (dF/dt max ) and the minimal velocity of relaxation (dF/dt min ) were normalized to the amplitude of developed force (Force peak ) at 0.7 and 2.0 mM extracellular Ca 2+ . Blood glucose levels were higher in STZ rats than in Ctr rats. STZ rats exhibited reduced Force peak , smaller dF/dt max /Force peak , smaller dF/dt min /Force peak , and a lower peak of intracellular Ca 2+ transients with a slower decay compared with Ctr rats. Both mitochondrial calcium uniporter (MCU) expression and MitoSOX Red fluorescence were elevated in STZ rats. In STZ rats, Ru360, an MCU inhibitor, decreased rhod-2 and MitoSOX Red fluorescence and increased both dF/dt max /Force peak and dF/dt min /Force peak . In contrast, spermine increased rhod-2 fluorescence but decreased dF/dt max /Force peak and dF/dt min /Force peak . Conclusions . Mitochondrial Ca 2+ uptake modulates myocardial contractile properties through altered ROS production at a relatively early stage of diabetes. mitochondrial calcium ROS myocardium streptozotocin diabetes mellitus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Patients with diabetes mellitus (DM) frequently develop heart failure due to impaired relaxation [ 22 , 33 ]. In these patients, mitochondrial dysfunction in the hearts [ 23 ] leads to contractile dysfunction [ 21 ], as mitochondria play central roles in regulating cardiac muscle contractility [ 14 , 22 ]. Mitochondria supply approximately 90% of the ATP required for cardiac contraction [ 18 , 40 ], and mitochondrial Ca 2+ is a key regulator of ATP production [ 13 ] through activation of dehydrogenase in the electron transport chain [ 2 ]. Many studies have reported reduced mitochondrial Ca 2+ levels in cardiomyocytes from diabetic hearts [ 8 , 15 ], which have been attributed to decreased expression of the mitochondrial calcium uniporter (MCU) [ 8 , 9 , 35 ] and increased expression of the mitochondrial sodium/calcium exchanger (NCLX), a major pathway for mitochondrial Ca 2+ efflux [ 37 ]. In contrast, other studies have reported increased mitochondrial Ca 2+ in electrically stimulated cardiomyocytes isolated from streptozotocin (STZ)-induced diabetic hearts [ 6 ], as well as in left ventricular (LV) myocardium from diabetic hearts subjected to phenylephrine-induced pressure overload [ 28 ]. To date, however, it remains unclear how mitochondrial Ca 2+ uptake contributes to contractile function in diabetic hearts. Mitochondria are a major source of reactive oxygen species (ROS) in cardiac muscle [ 27 ]. Both reduced mitochondrial Ca 2+ levels and excessive mitochondrial Ca 2+ accumulation have been shown to increases ROS production [ 16 ]. ROS production is enhanced by hyperglycemia [ 12 ] and is elevated in diabetic hearts [ 3 , 36 ]. Increased ROS can activate Ca 2+ release channels (RyRs) in the sarcoplasmic reticulum (SR), thereby promoting SR Ca 2+ leak [ 40 ]. However, the role of ROS in determining contractile function in diabetic myocardium has not yet been fully elucidated. Therefore, in the present study we focused on the contractile properties of diabetic myocardium and investigated how mitochondrial Ca 2+ modulates contraction and relaxation through alterations in ROS production using trabeculae obtained from diabetic rat hearts. MATERIALS and METHODS (See expanded Methods in the online data supplement) Animal model All animal procedures were approved by the Ethics Review Board of Tohoku University (approval reference number: 2019MdA-026-09). Sprague-Dawley rats weighing 180 g received a single subcutaneous injection of 55 mg/kg streptozotocin (STZ rats) or an equal volume of vehicle (Ctr rats) [ 11 ]. Experiments were performed six weeks after injection. Measurements of force and fluorescence signals in rat trabeculae Rats were adequately anesthetized by intraperitoneal injection of butorphanol tartrate (2.5 mg/kg), midazolam (2 mg/kg), and medetomidine chloride (0.15 mg/kg), and blood glucose levels were measured. The heart was then excised for sample preparation and weighed. Removal of the heart for dissection under sufficiently deep anesthesia resulted in euthanasia. Trabeculae were dissected from the right ventricle as previously described [ 26 , 31 ]. Force was measured using a strain gauge, and intracellular Ca 2+ was assessed using fura-2 [ 26 , 31 ]. Mitochondrial ROS production was estimated using MitoSOX Red fluorescence [ 1 , 32 ], and mitochondrial Ca 2+ levels were assessed using rhod-2 fluorescence [ 4 , 32 ]. The mitochondria were identified using MitoTracker Green [ 5 , 34 ] and confocal microscopy [ 32 ]. Contractile kinetics were evaluated as the maximal contraction velocity (dF/dt max ) and minimal relaxation velocity (dF/dt min ). To minimize the influence of differences in force amplitude, these parameters were normalized to peak force (Force peak ) [ 26 ]. MCU expression levels were determined using Western blot analysis [ 26 ]. Experimental protocol Force, fura-2 fluorescence, rhod-2 fluorescence, and MitoSOX Red fluorescence were measured during electrical stimulation at 0.5 Hz. Measurements were performed at extracellular Ca 2+ concentrations ([Ca 2+ ] o ) of 0.7 and 2.0 mM to evaluate Ca 2+ -dependent effects. To minimize motion artifacts during contraction, fluorescence signals were recorded immediately before electrical stimulation. To investigate the role of mitochondrial Ca 2+ uptake, force, rhod-2 fluorescence, and MitoSOX Red fluorescence were measured after the addition of 5 µM Ru360 [ 10 , 25 ] and 50 µM spermine [ 24 ] Statistics Data were presented as mean SEM. Statistical analyses were performed using one-way repeated-measures ANOVA followed by a post-hoc Tukey-Kramer test for multiple comparisons, and a t -test for two-group comparisons when the data were normally distributed, unless otherwise specified. Statistical analyses were performed using statistical analysis software (Ekuseru-Toukei, Social Survey Research Information Co., Ltd, Tokyo, Japan). A value of p < 0.05 was considered statistically significant. RESULTS Physical characteristics and MCU expression Blood glucose levels were higher in STZ rats than in Ctr rats (Fig. 1 A). Body weight and heart weight were lower in STZ rats than in Ctr rats (Fig. 1 B), although tibial length did not differ between the two groups (Fig. 1 C). MCU expression levels were measured in cardiac muscle from STZ and Ctr rats (Fig. 1 D). Surprisingly, the ratio of MCU to β-actin was higher in STZ rats than in Ctr rats (Fig. 1 E). Contractile properties and mitochondrial Ca 2+ STZ rats showed lower peak amplitude and a slower decay of intracellular Ca 2+ transients during electrical stimulation at 0.5 Hz (Fig. 2 A and 2 B). Consistent with these findings, STZ rats exhibited lower Force peak and smaller dF/dt max and dF/dt min (Fig. 2 C and S1A). After normalization of contraction and relaxation velocities to Force peak , both dF/dt max /Force peak and dF/dt min /Force peak remained smaller in STZ rats than in Ctr rats (Fig. 2 D and S1B). Role of mitochondrial Ca uptake in contractile properties To examine the contribution of mitochondrial Ca 2+ uptake to contraction and relaxation kinetics, mitochondrial Ca 2+ uptake was inhibited with Ru360 and stimulated with spermine. In STZ rats, normalized contraction and relaxation velocities were improved by Ru360 and impaired by spermine at 0.7 and 2.0 mM [Ca 2+ ] o (Fig. 3 A). In contrast, neither Ru360 nor spermine affected normalized velocities in Ctr rats (Fig. 3 B). To investigate whether these functional changes were associated with alterations in mitochondrial Ca 2+ levels, rhod-2 fluorescence was used as an indicator of mitochondrial Ca 2+ . The spatial distribution of rhod-2 fluorescence was similar to that of MitoTracker Green, indicating that rhod-2 was localized to the mitochondria (Fig. 4 A). In STZ rats, rhod-2 fluorescence was decreased in the presence of Ru360 and was restored by spermine at both 0.7 and 2.0 mM [Ca 2+ ] o , whereas no significant changes were observed in Ctr rats (Fig. 4 B and 4 C). Role of mitochondrial Ca uptake in ROS production To assess the effect of mitochondrial Ca 2+ uptake on ROS production, trabeculae were loaded with MitoSOX Red. The spatial fluorescence pattern of MitoSOX Red was similar to that of MitoTracker Green, indicating that MitoSOX Red was localized to the mitochondria (Fig. 5 A). Following the addition of H 2 O 2 , MitoSOX Red fluorescence increased and reached a plateau during electrical stimulation at 0.5 Hz (Fig. 5 B and 5 C). The ratio of MitoSOX Red fluorescence before H 2 O 2 addition (Fl) to that after H 2 O 2 addition (Fl H2O2 ) was calculated. This ratio was higher in STZ rats than in Ctr rats (Fig. 5 D), indicating elevated basal ROS production in STZ rats. Furthermore, Ru360 reduced MitoSOX Red fluorescence in STZ rats, but not in Ctr rats, at 0.7 and 2.0 mM [Ca 2+ ] o (Fig. 5 E), suggesting that mitochondrial Ca 2+ uptake contributes to enhanced ROS production in STZ rats. DISCUSSION The present study investigated how mitochondrial Ca 2+ uptake influences contractile properties in cardiac muscle from STZ-induced diabetic rats. To the best of our knowledge, this is the first study to demonstrate that, at a relatively early stage of diabetes, mitochondrial Ca 2+ uptake modulates myocardial contractile properties through alterations in mitochondrial ROS production in STZ rats. Contractile properties, ROS production, and MCU expression Past studies have shown that STZ rats exhibit reduced peak force, slower contraction and relaxation, and decreased Ca 2+ transient amplitude with prolonged decay in LV trabeculae [ 17 , 39 ] and isolated single myocytes [ 29 ]. The findings of the present study are consistent with these reports (Fig. 2 ). Furthermore, mitochondrial ROS production was higher in STZ rats than in Ctr rats (Fig. 5 D), in agreement with past studies demonstrating elevated ROS levels in diabetic hearts [ 3 , 36 ]. In contrast to several past studies reporting reduced MCU expression in diabetic myocardium [ 8 , 9 , 35 ], we observed increased MCU expression in STZ rat hearts (Fig. 1 E). This discrepancy may be explained, at least in part, by differences in disease stage. Our experiments were performed at a relatively early stage of diabetes, before the development of overt heart failure or severe mitochondrial damage. Under these conditions, upregulation of MCU may represent a compensatory response to preserve mitochondrial Ca²⁺ uptake [ 13 ] and sustain ATP production through Ca²⁺-dependent activation of mitochondrial dehydrogenases [ 2 ] during diabetic metabolic stress. Taken together, these findings suggest that MCU expression is upregulated during the early phase of diabetes, before the development of overt heart failure, and that this increase may represent a compensatory response that contributes to impaired myocardial contractile function observed in Fig. 2 . Role of mitochondrial Ca 2+ uptake in contractile properties Mitochondrial Ca 2+ plays an essential role in ATP production [ 13 ] by activating dehydrogenase in the electron transport chain [ 2 ]. However, excessive mitochondrial Ca 2+ accumulation has been shown to impair ATP production [ 20 ] and induce cell death [ 30 ], indicating that an optimal mitochondrial Ca 2+ concentration level is require for normal cardiac contraction. In the present study, inhibition of mitochondrial Ca 2+ uptake with Ru360 decreased mitochondrial Ca 2+ levels and improved contractile properties, whereas stimulation of mitochondrial Ca 2+ uptake with spermine increased mitochondrial Ca 2+ levels and impaired contractile properties in STZ rats (Fig. 3 A and 4 C). These findings suggest that excessive mitochondrial Ca 2+ accumulation contributes to contractile dysfunction in STZ rats. Regarding the role of mitochondrial Ca 2+ in ROS production, Hamilton et al. reported that complete restoration of mitochondrial Ca 2+ increases mitochondrial ROS production, whereas partial restoration reduces it in hypertrophic rat myocytes [ 16 ], suggesting that a complex relationship between mitochondrial Ca 2+ and ROS production. In the present study, inhibition of mitochondrial Ca 2+ uptake by Ru360 decreased ROS production in STZ rats (Fig. 5 E). ROS are known to activate sarcoplasmic reticulum (SR) Ca 2+ release channels [ 40 ] and increase Ca 2+ spark frequency from the SR [ 38 ]. Increased Ca 2+ spark frequency elevates diastolic Ca 2+ concentration [ 19 ], which leads to contractile dysfunction [ 7 ]. Therefore, it is reasonable to assume that, in STZ rats, inhibition of mitochondrial Ca 2+ uptake reduces mitochondrial Ca 2+ toward a more optimal range, thereby reducing mitochondrial ROS production. The resulting decrease in ROS may improve contractile function by suppressing excessive SR Ca 2+ release. Limitations The STZ-induced diabetes model is generally considered to represent type 1 DM. In addition, the measurements were performed under nonphysiological conditions, that is, at room temperature and with electrical stimulation at 0.5 Hz. Therefore, caution is required when interpreting the results of the present study. Declarations Availability of data and materials The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethical approval All animal procedures were approved by the Ethics Review Board of Tohoku University (approval reference number: 2019MdA-026-09). Consent for publication All authors have read the manuscript and given explicit consent to submit. All authors are completely satisfied with its publication. Author information Author ORCIDs Masahito Miura ://orcid.org/0000-0003-0313-1308 Author contributions Conceptualization: HS, MM Formal analysis: HS, SK Funding acquisition: HS, MM Investigation: HS, SK, AM, SA Methodology: HS, AM, KT Project administration: KY, TO Resources: MM Supervision: MM Validation: HS, SA, KT Visualization: HS, TO, MM Writing– original draft: HS, MM Competing interest The authors declare that there are no competing interests. Funding This work was supported by Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (M. Miura, No 2019MdA-026-09). Acknowledgements Not applicable. References Andersson DC, Fauconnier J, Yamada T, Lacampagne A, Zhang SJ, Katz A, Westerblad H (2011) Mitochondrial production of reactive oxygen species contributes to the β-adrenergic stimulation of mouse cardiomyocytes. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9025606","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":605938301,"identity":"7d209fa5-18e2-4847-93a0-6edade6e6fb9","order_by":0,"name":"Haruka Sato","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Haruka","middleName":"","lastName":"Sato","suffix":""},{"id":605938314,"identity":"57e24828-eb14-4ca7-946d-ec93e4af6c20","order_by":1,"name":"Sana Koyama","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sana","middleName":"","lastName":"Koyama","suffix":""},{"id":605938319,"identity":"b5ee1d4c-5811-4bed-b318-4345bdf66023","order_by":2,"name":"Ayana Matsumoto","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ayana","middleName":"","lastName":"Matsumoto","suffix":""},{"id":605938321,"identity":"fcb184cb-a38b-4e3d-99ee-621b9ad7964a","order_by":3,"name":"Shiori Akazawa","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shiori","middleName":"","lastName":"Akazawa","suffix":""},{"id":605938322,"identity":"43e11f03-80c7-4d1d-b544-7ca40d9acaae","order_by":4,"name":"Kao Takeda","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kao","middleName":"","lastName":"Takeda","suffix":""},{"id":605938323,"identity":"e36080bf-afdf-4241-bb83-04c84b795b7c","order_by":5,"name":"Karin Yamamoto","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Karin","middleName":"","lastName":"Yamamoto","suffix":""},{"id":605938326,"identity":"54f3f9fd-db2e-492b-acae-a63b182dd954","order_by":6,"name":"Tsuyoshi Okumura","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tsuyoshi","middleName":"","lastName":"Okumura","suffix":""},{"id":605938328,"identity":"04c0a57c-f67a-48e2-886d-d62128f5ae0b","order_by":7,"name":"Masahito Miura","email":"data:image/png;base64,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","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Masahito","middleName":"","lastName":"Miura","suffix":""}],"badges":[],"createdAt":"2026-03-04 04:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9025606/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9025606/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104789914,"identity":"67252bcd-530e-44ed-a997-364b729cbb05","added_by":"auto","created_at":"2026-03-17 08:31:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysical characteristics and MCU expression in Ctr and STZ rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Blood glucose levels in control (Ctr) (n = 6) rats and streptozotocin (STZ) rats (n = 9). **p\u0026lt;0.01 vs Ctr\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Body weight (left panel) and heart weight (right panel) in Ctr (n = 6) rats and STZ rats (n = 9). **p\u0026lt;0.01 vs Ctr; *p\u0026lt;0.05 vs Ctr\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Tibial length in Ctr (n = 7) rats and STZ rats (n = 10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Representative Western blots of MCU and b-actin in hearts from Ctr rats and STZ rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Ratio of MCU to b-actin in hearts from Ctr (n = 6) rats and STZ rats (n = 6). **p\u0026lt;0.005 vs Ctr\u003c/p\u003e","description":"","filename":"STZFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9025606/v1/f6e2a5e2e95125fca46d59c7.png"},{"id":104790138,"identity":"bd563541-3cf6-4976-a579-882c8cb5720b","added_by":"auto","created_at":"2026-03-17 08:32:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCa\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e transients and force in Ctr and STZ rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative recordings of Ca\u003csup\u003e2+\u003c/sup\u003e transients (upper) and force (lower) at 0.7 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e in Ctr rats and STZ rats during electrical stimulation at 0.5 Hz. ST indicates the moment of electrical stimulation. Experimental number 171024 and 171101.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Summary data for diastolic [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e (left), peak [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e (middle), and the time constant of [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e decline (right) of Ca\u003csup\u003e2+\u003c/sup\u003e transients at 0.7 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e in Ctr rats (n = 6) and STZ rats (n = 9). **p\u0026lt;0.01 vs Ctr; *p\u0026lt;0.05 vs Ctr\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Summary data for peak force (Force\u003csub\u003epeak\u003c/sub\u003e, left), maximal contraction velocity (dF/dt\u003csub\u003emax\u003c/sub\u003e, middle), and minimal relaxation velocity (dF/dt\u003csub\u003emin\u003c/sub\u003e, right) at 0.7 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e in Ctr rats (n = 6) and STZ rats (n = 9). **p\u0026lt;0.01 vs Ctr; *p\u0026lt;0.05 vs Ctr\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Contraction and relaxation velocities normalized to peak force (Force\u003csub\u003epeak\u003c/sub\u003e). The left panel shows representative recordings of force normalized to their respective Force\u003csub\u003epeak\u003c/sub\u003e values. The middle and right panels show summary data for normalized contraction velocity (dF/dt\u003csub\u003emax\u003c/sub\u003e) and relaxation velocity (dF/dt\u003csub\u003emin\u003c/sub\u003e), respectively, at 0.7 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e in Ctr rats (n = 6) and STZ rats (n = 9), respectively. *p\u0026lt;0.05 vs Ctr\u003c/p\u003e","description":"","filename":"STZFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9025606/v1/48e926ede6eb6b1c7eb892ef.png"},{"id":104789199,"identity":"c896a3b4-145a-4bb3-9c73-aad7bc51a3c9","added_by":"auto","created_at":"2026-03-17 08:28:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRoles of mitochondrial Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e uptake in contraction and relaxation properties in Ctr and STZ rat hearts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e The left panel shows representative recordings of force normalized to their respective Force\u003csub\u003epeak\u003c/sub\u003e in the absence and presence of Ru360 and spermine in STZ rats (Experimental number 230406). The middle and right panels show summary data for the effects of Ru360 and spermine on normalized dF/dt\u003csub\u003emax\u003c/sub\u003e (upper) and normalized dF/dt\u003csub\u003emin\u003c/sub\u003e (lower) at 0.7 (middle, n = 6) and 2.0 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e (right, n = 7) in STZ rats. Ru indicates Ru360, and Sp indicates spermine. **p\u0026lt;0.01 vs Ru; *p\u0026lt;0.05 vs Ru\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e The left panel shows representative recordings of force normalized to their respective Force\u003csub\u003epeak\u003c/sub\u003e in the absence and presence of Ru360 and spermine in Ctr rats (Experimental number 230724). The middle and right panels show summary data for the effects of Ru360 and spermine on normalized dF/dt\u003csub\u003emax\u003c/sub\u003e (upper) and normalized dF/dt\u003csub\u003emin\u003c/sub\u003e (lower) at 0.7 (middle, n = 5) and 2.0 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e (right, n = 5) in Ctr rats.\u003c/p\u003e","description":"","filename":"STZFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9025606/v1/c742ce762f38fbc844e0bd06.png"},{"id":104789766,"identity":"03d73256-ae94-4b4a-be41-c376d4c98008","added_by":"auto","created_at":"2026-03-17 08:31:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":145775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRoles of mitochondrial Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e uptake in rhod-2 fluorescence in Ctr and STZ rat hearts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative confocal images of a trabecula loaded with MitoTracker Green (left) and rhod-2 (middle). The right panel shows the merged image.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Representative recordings of force (upper) and rhod-2 fluorescence (lower) in the absence (red) and presence of Ru360 (blue) in STZ rats (Experimental number 221118). Changes in rhod-2 fluorescence were calculated using the signal immediately before electrical stimulation, indicated by the grey box. ST indicates the moment of electrical stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Summary data for the effects of Ru360 and spermine on rhod-2 fluorescence at 0.7 mM (upper) and 2.0 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e (lower) in STZ rats (n =4, left) and Ctr rats (n =4, right). Ru indicates Ru360, and Sp indicates spermine. **p\u0026lt;0.01 vs Ru; *p\u0026lt;0.05 vs Ru\u003c/p\u003e","description":"","filename":"STZFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9025606/v1/e2fc854574f0177e7a498abd.png"},{"id":104789777,"identity":"5d031b5e-9f14-4cf2-94d6-1cfe4d716e9b","added_by":"auto","created_at":"2026-03-17 08:31:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":217352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitoSOX Red fluorescence in Ctr and STZ rat hearts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative confocal images of a trabecula loaded with MitoTracker Green (left) and MitoSOX Red (middle). The right panel shows the merged image.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Representative recording of MitoSOX Red fluorescence before (Fl) and after the addition of 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fl\u003csub\u003eH2O2\u003c/sub\u003e) in a trabecula from a STZ rat at 2 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e (Ex 230728).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Expanded recordings of MitoSOX Red fluorescence (upper) and force (lower) during the periods indicated by lines (a) (left) and (b) (right) in panel B. ST indicates the moment of electrical stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Summary data for the ratio of Fl to Fl\u003csub\u003eH2O2\u003c/sub\u003e in Ctr rats (n = 4) and STZ rats (n = 3) at 2 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e. *p\u0026lt;0.05 vs Ctr\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Summary data for the effect of Ru360 on MitoSOX Red fluorescence in STZ rats (n = 5, left) and Ctr rats (n = 5, right) at 0.7 mM (upper) and 2 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e (lower). Ru indicates Ru360. *p\u0026lt;0.05 vs (-)\u003c/p\u003e","description":"","filename":"STZFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9025606/v1/0f0f04809ce0a3306ae0f767.png"},{"id":105564053,"identity":"597fe7f5-0e10-4495-9f8a-8d711f26ed28","added_by":"auto","created_at":"2026-03-27 12:48:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1424578,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9025606/v1/c91d7793-95f6-487d-92e7-b81fcf734365.pdf"},{"id":104790071,"identity":"201b9c0a-c1b6-4689-881c-9abeea05f3b3","added_by":"auto","created_at":"2026-03-17 08:32:34","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":715903,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinemanuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-9025606/v1/4c24209367d2f86e342c4f4d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial Ca 2+ uptake affects contractile properties in streptozotocin-induced diabetic rat myocardium","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePatients with diabetes mellitus (DM) frequently develop heart failure due to impaired relaxation [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. In these patients, mitochondrial dysfunction in the hearts [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e] leads to contractile dysfunction [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], as mitochondria play central roles in regulating cardiac muscle contractility [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Mitochondria supply approximately 90% of the ATP required for cardiac contraction [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e], and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e is a key regulator of ATP production [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e] through activation of dehydrogenase in the electron transport chain [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. Many studies have reported reduced mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels in cardiomyocytes from diabetic hearts [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e], which have been attributed to decreased expression of the mitochondrial calcium uniporter (MCU) [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e] and increased expression of the mitochondrial sodium/calcium exchanger (NCLX), a major pathway for mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e efflux [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. In contrast, other studies have reported increased mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e in electrically stimulated cardiomyocytes isolated from streptozotocin (STZ)-induced diabetic hearts [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e], as well as in left ventricular (LV) myocardium from diabetic hearts subjected to phenylephrine-induced pressure overload [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. To date, however, it remains unclear how mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake contributes to contractile function in diabetic hearts.\u003c/p\u003e \u003cp\u003eMitochondria are a major source of reactive oxygen species (ROS) in cardiac muscle [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Both reduced mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels and excessive mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e accumulation have been shown to increases ROS production [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. ROS production is enhanced by hyperglycemia [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e] and is elevated in diabetic hearts [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Increased ROS can activate Ca\u003csup\u003e2+\u003c/sup\u003e release channels (RyRs) in the sarcoplasmic reticulum (SR), thereby promoting SR Ca\u003csup\u003e2+\u003c/sup\u003e leak [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, the role of ROS in determining contractile function in diabetic myocardium has not yet been fully elucidated.\u003c/p\u003e \u003cp\u003eTherefore, in the present study we focused on the contractile properties of diabetic myocardium and investigated how mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e modulates contraction and relaxation through alterations in ROS production using trabeculae obtained from diabetic rat hearts.\u003c/p\u003e "},{"header":"MATERIALS and METHODS","content":"\u003cp\u003e (See expanded Methods in the online data supplement)\u003c/p\u003e\u003ch3\u003eAnimal model\u003c/h3\u003e\u003cp\u003e All animal procedures were approved by the Ethics Review Board of Tohoku University (approval reference number: 2019MdA-026-09). Sprague-Dawley rats weighing 180 g received a single subcutaneous injection of 55 mg/kg streptozotocin (STZ rats) or an equal volume of vehicle (Ctr rats) [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. Experiments were performed six weeks after injection.\u003c/p\u003e\u003ch2\u003eMeasurements of force and fluorescence signals in rat trabeculae\u003c/h2\u003e\u003cp\u003eRats were adequately anesthetized by intraperitoneal injection of butorphanol tartrate (2.5 mg/kg), midazolam (2 mg/kg), and medetomidine chloride (0.15 mg/kg), and blood glucose levels were measured. The heart was then excised for sample preparation and weighed. Removal of the heart for dissection under sufficiently deep anesthesia resulted in euthanasia. Trabeculae were dissected from the right ventricle as previously described [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Force was measured using a strain gauge, and intracellular Ca\u003csup\u003e2+\u003c/sup\u003e was assessed using fura-2 [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Mitochondrial ROS production was estimated using MitoSOX Red fluorescence [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e], and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels were assessed using rhod-2 fluorescence [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. The mitochondria were identified using MitoTracker Green [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e] and confocal microscopy [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Contractile kinetics were evaluated as the maximal contraction velocity (dF/dt\u003csub\u003emax\u003c/sub\u003e) and minimal relaxation velocity (dF/dt\u003csub\u003emin\u003c/sub\u003e). To minimize the influence of differences in force amplitude, these parameters were normalized to peak force (Force\u003csub\u003epeak\u003c/sub\u003e) [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. MCU expression levels were determined using Western blot analysis [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003ch3\u003eExperimental protocol\u003c/h3\u003e\u003cp\u003eForce, fura-2 fluorescence, rhod-2 fluorescence, and MitoSOX Red fluorescence were measured during electrical stimulation at 0.5 Hz. Measurements were performed at extracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentrations ([Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e) of 0.7 and 2.0 mM to evaluate Ca\u003csup\u003e2+\u003c/sup\u003e-dependent effects. To minimize motion artifacts during contraction, fluorescence signals were recorded immediately before electrical stimulation. To investigate the role of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake, force, rhod-2 fluorescence, and MitoSOX Red fluorescence were measured after the addition of 5 µM Ru360 [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] and 50 µM spermine [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003ch3\u003eStatistics\u003c/h3\u003e\u003cp\u003eData were presented as mean SEM. Statistical analyses were performed using one-way repeated-measures ANOVA followed by a post-hoc Tukey-Kramer test for multiple comparisons, and a \u003cem\u003et\u003c/em\u003e-test for two-group comparisons when the data were normally distributed, unless otherwise specified. Statistical analyses were performed using statistical analysis software (Ekuseru-Toukei, Social Survey Research Information Co., Ltd, Tokyo, Japan). A value of p \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePhysical characteristics and MCU expression\u003c/h2\u003e \u003cp\u003eBlood glucose levels were higher in STZ rats than in Ctr rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Body weight and heart weight were lower in STZ rats than in Ctr rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), although tibial length did not differ between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMCU expression levels were measured in cardiac muscle from STZ and Ctr rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Surprisingly, the ratio of MCU to β-actin was higher in STZ rats than in Ctr rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eContractile properties and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eSTZ rats showed lower peak amplitude and a slower decay of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e transients during electrical stimulation at 0.5 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Consistent with these findings, STZ rats exhibited lower Force\u003csub\u003epeak\u003c/sub\u003e and smaller dF/dt\u003csub\u003emax\u003c/sub\u003e and dF/dt\u003csub\u003emin\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and S1A). After normalization of contraction and relaxation velocities to Force\u003csub\u003epeak\u003c/sub\u003e, both dF/dt\u003csub\u003emax\u003c/sub\u003e /Force\u003csub\u003epeak\u003c/sub\u003e and dF/dt\u003csub\u003emin\u003c/sub\u003e /Force\u003csub\u003epeak\u003c/sub\u003e remained smaller in STZ rats than in Ctr rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and S1B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRole of mitochondrial Ca uptake in contractile properties\u003c/h3\u003e\n\u003cp\u003eTo examine the contribution of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake to contraction and relaxation kinetics, mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake was inhibited with Ru360 and stimulated with spermine. In STZ rats, normalized contraction and relaxation velocities were improved by Ru360 and impaired by spermine at 0.7 and 2.0 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In contrast, neither Ru360 nor spermine affected normalized velocities in Ctr rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether these functional changes were associated with alterations in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels, rhod-2 fluorescence was used as an indicator of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e. The spatial distribution of rhod-2 fluorescence was similar to that of MitoTracker Green, indicating that rhod-2 was localized to the mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In STZ rats, rhod-2 fluorescence was decreased in the presence of Ru360 and was restored by spermine at both 0.7 and 2.0 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e, whereas no significant changes were observed in Ctr rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRole of mitochondrial Ca uptake in ROS production\u003c/h3\u003e\n\u003cp\u003eTo assess the effect of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake on ROS production, trabeculae were loaded with MitoSOX Red. The spatial fluorescence pattern of MitoSOX Red was similar to that of MitoTracker Green, indicating that MitoSOX Red was localized to the mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Following the addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MitoSOX Red fluorescence increased and reached a plateau during electrical stimulation at 0.5 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The ratio of MitoSOX Red fluorescence before H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e addition (Fl) to that after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e addition (Fl\u003csub\u003eH2O2\u003c/sub\u003e) was calculated. This ratio was higher in STZ rats than in Ctr rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), indicating elevated basal ROS production in STZ rats. Furthermore, Ru360 reduced MitoSOX Red fluorescence in STZ rats, but not in Ctr rats, at 0.7 and 2.0 mM [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), suggesting that mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake contributes to enhanced ROS production in STZ rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study investigated how mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake influences contractile properties in cardiac muscle from STZ-induced diabetic rats. To the best of our knowledge, this is the first study to demonstrate that, at a relatively early stage of diabetes, mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake modulates myocardial contractile properties through alterations in mitochondrial ROS production in STZ rats.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eContractile properties, ROS production, and MCU expression\u003c/h2\u003e \u003cp\u003ePast studies have shown that STZ rats exhibit reduced peak force, slower contraction and relaxation, and decreased Ca\u003csup\u003e2+\u003c/sup\u003e transient amplitude with prolonged decay in LV trabeculae [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and isolated single myocytes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The findings of the present study are consistent with these reports (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, mitochondrial ROS production was higher in STZ rats than in Ctr rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), in agreement with past studies demonstrating elevated ROS levels in diabetic hearts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast to several past studies reporting reduced MCU expression in diabetic myocardium [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], we observed increased MCU expression in STZ rat hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). This discrepancy may be explained, at least in part, by differences in disease stage. Our experiments were performed at a relatively early stage of diabetes, before the development of overt heart failure or severe mitochondrial damage. Under these conditions, upregulation of MCU may represent a compensatory response to preserve mitochondrial Ca\u0026sup2;⁺ uptake [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and sustain ATP production through Ca\u0026sup2;⁺-dependent activation of mitochondrial dehydrogenases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] during diabetic metabolic stress.\u003c/p\u003e \u003cp\u003eTaken together, these findings suggest that MCU expression is upregulated during the early phase of diabetes, before the development of overt heart failure, and that this increase may represent a compensatory response that contributes to impaired myocardial contractile function observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRole of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake in contractile properties\u003c/h2\u003e \u003cp\u003eMitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e plays an essential role in ATP production [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] by activating dehydrogenase in the electron transport chain [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, excessive mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e accumulation has been shown to impair ATP production [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and induce cell death [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], indicating that an optimal mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration level is require for normal cardiac contraction. In the present study, inhibition of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake with Ru360 decreased mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels and improved contractile properties, whereas stimulation of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake with spermine increased mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels and impaired contractile properties in STZ rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These findings suggest that excessive mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e accumulation contributes to contractile dysfunction in STZ rats.\u003c/p\u003e \u003cp\u003eRegarding the role of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e in ROS production, Hamilton et al. reported that complete restoration of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e increases mitochondrial ROS production, whereas partial restoration reduces it in hypertrophic rat myocytes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], suggesting that a complex relationship between mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e and ROS production. In the present study, inhibition of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake by Ru360 decreased ROS production in STZ rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). ROS are known to activate sarcoplasmic reticulum (SR) Ca\u003csup\u003e2+\u003c/sup\u003e release channels [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and increase Ca\u003csup\u003e2+\u003c/sup\u003e spark frequency from the SR [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Increased Ca\u003csup\u003e2+\u003c/sup\u003e spark frequency elevates diastolic Ca\u003csup\u003e2+\u003c/sup\u003e concentration [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], which leads to contractile dysfunction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, it is reasonable to assume that, in STZ rats, inhibition of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake reduces mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e toward a more optimal range, thereby reducing mitochondrial ROS production. The resulting decrease in ROS may improve contractile function by suppressing excessive SR Ca\u003csup\u003e2+\u003c/sup\u003e release.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThe STZ-induced diabetes model is generally considered to represent type 1 DM. In addition, the measurements were performed under nonphysiological conditions, that is, at room temperature and with electrical stimulation at 0.5 Hz. Therefore, caution is required when interpreting the results of the present study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Ethics Review Board of Tohoku University (approval reference number: 2019MdA-026-09).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read the manuscript and\u0026nbsp;given explicit consent to submit. All authors are completely satisfied with its publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor ORCIDs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMasahito Miura ://orcid.org/0000-0003-0313-1308\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization: HS, MM\u003c/p\u003e\n\u003cp\u003eFormal analysis: HS, SK\u003c/p\u003e\n\u003cp\u003eFunding acquisition: HS, MM\u003c/p\u003e\n\u003cp\u003eInvestigation: HS, SK, AM, SA\u003c/p\u003e\n\u003cp\u003eMethodology: HS, AM, KT\u003c/p\u003e\n\u003cp\u003eProject administration: KY, TO\u003c/p\u003e\n\u003cp\u003eResources: MM\u003c/p\u003e\n\u003cp\u003eSupervision: MM\u003c/p\u003e\n\u003cp\u003eValidation: HS, SA, KT\u003c/p\u003e\n\u003cp\u003eVisualization: HS, TO, MM\u003c/p\u003e\n\u003cp\u003eWriting– original draft: HS, MM\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (M. Miura, No 2019MdA-026-09).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndersson DC, Fauconnier J, Yamada T, Lacampagne A, Zhang SJ, Katz A, Westerblad H (2011) Mitochondrial production of reactive oxygen species contributes to the β-adrenergic stimulation of mouse cardiomyocytes. J Physiol 589:1791\u0026ndash;1801. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1113/jphysiol.2010.202838\u003c/span\u003e\u003cspan address=\"10.1113/jphysiol.2010.202838\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalaban RS (2012) Perspectives on: SGP symposium on mitochondrial physiology and medicine: metabolic homeostasis of the heart. 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Cardiovasc Res 77:432\u0026ndash;441. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/cvr/cvm047\u003c/span\u003e\u003cspan address=\"10.1093/cvr/cvm047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Cannell MB, Phillips AR, Cooper GJ, Ward ML (2008) Altered calcium homeostasis does not explain the contractile deficit of diabetic cardiomyopathy. Diabetes 57:2158\u0026ndash;2166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2337/db08-0140\u003c/span\u003e\u003cspan address=\"10.2337/db08-0140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZima AV, Blatter LA (2006) Redox regulation of cardiac calcium channels and transporters. Cardiovasc Res 71:310\u0026ndash;321. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cardiores.2006.02.019\u003c/span\u003e\u003cspan address=\"10.1016/j.cardiores.2006.02.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mitochondrial calcium, ROS, myocardium, streptozotocin, diabetes mellitus","lastPublishedDoi":"10.21203/rs.3.rs-9025606/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9025606/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eDiabetes mellitus is a major risk factor for the development of heart failure. However, the role of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e in regulating contractile function in diabetic myocardium has not been fully elucidated. In the present study, we examined how mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake influences myocardial contractile properties under diabetic conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Results:\u003c/strong\u003e Rats were injected with 55 mg/kg streptozotocin (STZ rats) or solvent (Ctr rats). Six weeks after the injection, trabeculae were dissected from the right ventricles. Force was recorded with a strain gauge, intracellular Ca\u003csup\u003e2+\u003c/sup\u003e with fura-2, reactive oxygen species (ROS) production with MitoSOX Red, and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e with rhod-2 in trabeculae. The maximal velocity of contraction (dF/dt\u003csub\u003emax\u003c/sub\u003e) and the minimal velocity of relaxation (dF/dt\u003csub\u003emin\u003c/sub\u003e) were normalized to the amplitude of developed force (Force\u003csub\u003epeak\u003c/sub\u003e) at 0.7 and 2.0 mM extracellular Ca\u003csup\u003e2+\u003c/sup\u003e. Blood glucose levels were higher in STZ rats than in Ctr rats. STZ rats exhibited reduced Force\u003csub\u003epeak\u003c/sub\u003e, smaller dF/dt\u003csub\u003emax\u003c/sub\u003e/Force\u003csub\u003epeak\u003c/sub\u003e, smaller dF/dt\u003csub\u003emin\u003c/sub\u003e/Force\u003csub\u003epeak\u003c/sub\u003e, and a lower peak of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e transients with a slower decay compared with Ctr rats. Both mitochondrial calcium uniporter (MCU) expression and MitoSOX Red fluorescence were elevated in STZ rats. In STZ rats, Ru360, an MCU inhibitor, decreased rhod-2 and MitoSOX Red fluorescence and increased both dF/dt\u003csub\u003emax\u003c/sub\u003e/Force\u003csub\u003epeak\u003c/sub\u003e and dF/dt\u003csub\u003emin\u003c/sub\u003e/Force\u003csub\u003epeak\u003c/sub\u003e. In contrast, spermine increased rhod-2 fluorescence but decreased dF/dt\u003csub\u003emax\u003c/sub\u003e/Force\u003csub\u003epeak\u003c/sub\u003e and dF/dt\u003csub\u003emin\u003c/sub\u003e/Force\u003csub\u003epeak\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e. Mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake modulates myocardial contractile properties through altered ROS production at a relatively early stage of diabetes.\u003c/p\u003e","manuscriptTitle":"Mitochondrial Ca 2+ uptake affects contractile properties in streptozotocin-induced diabetic rat myocardium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-17 08:08:57","doi":"10.21203/rs.3.rs-9025606/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"940ea98c-ce26-4155-a88d-dfc1c573a5a7","owner":[],"postedDate":"March 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T06:56:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-17 08:08:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9025606","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9025606","identity":"rs-9025606","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}