STIM1-dependent treg dysfunction promotes cardiometabolic HFpEF: insights from patients and animal studies

Abstract

Background

Heart failure with preserved ejection fraction (HFpEF) arises from chronic cardiometabolic and vascular stress and is increasingly recognized as an inflammatory syndrome with immune dysregulation. Regulatory T cells (Tregs) are critical modulators of cardiovascular inflammation, yet the mechanisms driving Treg dysfunction in HFpEF remain poorly defined. stromal interaction molecule 1 (STIM1)-dependent calcium signaling is a key stress-responsive pathway in immune cells; however, its role in Treg maladaptation during HFpEF remains unknown.

Methods

Circulating Tregs from patients with and without HFpEF were analyzed for abundance, STIM1 expression, and stress-associated signaling pathways. To establish causality, mice with Treg-specific deletion of STIM1 (Treg^Stim1-/-) and littermate controls were subjected to a high-fat diet and nitric oxide synthase inhibition (L-NAME) to induce a cardiometabolic HFpEF model. Cardiac diastolic function, vascular reactivity, blood pressure, and exercise capacity were assessed alongside structural remodeling.

Results

Patients with HFpEF exhibited reduced circulating Treg numbers accompanied by increased STIM1 expression and activation of apoptotic, inflammatory, and ER stress pathways, consistent with stress-induced Treg instability. In vivo, control mice developed features of HFpEF, including diastolic dysfunction with preserved ejection fraction, hypertension, metabolic dysregulation, endothelial dysfunction, cardiac fibrosis, and impaired exercise tolerance. In contrast, Treg^Stim1-/- mice were protected from these abnormalities. Mechanistically, STIM1 signaling promoted loss of Treg suppressive stability and the acquisition of effector-like inflammatory signaling, including IL-17- and IFN-γ-dependent cardiomyocyte activation, whereas STIM1-deficient Tregs maintained a non-pathogenic phenotype.

Conclusions

STIM1-dependent stress signaling drives maladaptive Treg instability that amplifies cardiovascular inflammation and HFpEF progression. These findings identify Treg STIM1 as a key driver of immune-mediated HFpEF progression and provide mechanistic evidence from humans to mice supporting immune-targeted therapeutic strategies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12933-026-03253-6.

Introduction

Heart failure with preserved ejection fraction (HFpEF) is a systemic multi-organ disorder, highlighting a major unmet need in cardiovascular medicine, with limited therapeutic options available. HFpEF prevalence is increasing and accounts for more than 50% of heart failure cases [1–4]. Thus, understanding the pathways and mechanisms involved in HFpEF pathogenesis is essential. With the possible exception of SGLT2I, almost all large-scale clinical trials aimed at improving HFpEF yielded neutral results [5, 6]. Therefore, there is a critical need to elucidate additional cellular and molecular mechanisms and to identify new drugable targets to rescue the HFpEF phenotype. A significant number of patients with HFpEF harbor the comorbidities of hypertension and obesity with type 2 diabetes [7, 8], which result in chronic cardio-metabolic-physical stress. As noted previously, many pharmacologic interventions tested in isolation fail to alter HFpEF outcomes, indicating that HFpEF likely arises from the convergence of multiple interacting pathological processes rather than a single dominant driver [8]. For example, conventional therapies targeting hypertension or diabetes alone do not consistently or robustly improve HFpEF-related pathophysiology [8].

Recent studies suggest that myocardial stiffness, inflammation, and microvascular endothelial dysfunction are central contributors to HFpEF development [9]. In addition, high mobility group protein B1 signaling and mitochondrial hyperacetylation have been implicated in HFpEF pathogenesis [10]. Central to the development of the HFpEF phenotype was the novel observation that the immune T cells contribute to hypertension-induced vascular dysfunction (angiotensin II-dependent) [11] and that transferring healthy immune regulatory CD4 + CD25+ cells (Treg) from control mice into mice infused with angiotensin II prevented hypertension and vascular endothelial dysfunction [12, 13]. Although HFpEF has not traditionally been conceptualized as an immune-mediated disease, these observations suggest that impaired immune regulation may contribute to cardiovascular vulnerability in conditions that predispose to HFpEF. Importantly, a preclinical mouse model of HFpEF that recapitulates many clinical features observed in patients has been developed, enabling investigation of how vascular dysfunction, hypertension, and metabolic stress interact to drive HFpEF pathogenesis [14].

Rather than attempting to map the contribution of immune cells to HFpEF de novo, the present study was motivated by the observation that HFpEF-relevant metabolic, inflammatory, and neurohumoral stressors converge on intracellular calcium handling and endoplasmic reticulum (ER) stress pathways. STIM is a key regulator of these processes and therefore represents a candidate integrator of stress signaling within immune cells. STIM1 is primarily localized to the ER, where it functions as a calcium sensor and regulates store-operated calcium entry (SOCE), a fundamental mechanism controlling intracellular calcium homeostasis in response to ER calcium depletion [15]. STIM1 proteins are ubiquitously expressed Ca²⁺ signal mediators that synchronize signaling and Ca²⁺ release from intracellular stores with control of Ca²⁺ entry channels in the plasma membrane [16–18]. STIM1 proteins are also essential for activating Orai1 and Orai3 channels, which are activated by store-independent intracellular actions of the inflammatory lipid second messenger, leukotriene C4 [19].

Previous studies have shown that STIM1 plays differential roles in endothelial cells, smooth muscle cells (SMCs), and cardiomyocytes. Conditional deletion of STIM1 in endothelial cells causes endothelial dysfunction [20], whereas its deletion in SMCs does not affect endothelial function [21]. However, the deletion or the overexpression of STIM1 in cardiomyocytes causes heart failure [22, 23]. These findings underscore the necessity of cell-specific approaches when evaluating STIM1 as a therapeutic target and highlight the potential for divergent outcomes depending on the cellular context in which STIM1 signaling is disrupted.

Additionally, it has been reported that Treg cell numbers are reduced in hypertension-related cardiovascular complications [12, 24, 25]. However, whether STIM1-dependent signaling in Treg cells contributes to HFpEF pathogenesis remains unexplored. Because STIM1 regulates both immune cell development and stress-adaptive calcium signaling, alterations in STIM1 expression within Treg cells may influence their survival, functional stability, and capacity to restrain inflammation under cardiometabolic stress. Accordingly, the present study sought to investigate the role of STIM1 in regulatory T (Treg) cells and to determine whether Treg-specific STIM1 deficiency modulates cardiovascular dysfunction associated with HFpEF.

To address this question, we utilized Stim1^flox/flox and Treg-specific Stim1^−/− male mice maintained on either a standard diet or a high-fat diet supplemented with L-NAME in drinking water for five weeks to induce HFpEF. In parallel, we analyzed blood samples from patients with and without HFpEF to quantify Treg cell abundance and assess STIM1 expression within these cells. Together, this combined experimental and clinical approach provides mechanistic insight into how STIM1-dependent Treg dysfunction may contribute to HFpEF-associated cardiovascular pathology and establishes translational relevance to human disease. The objective of this study was not to re-establish the involvement of regulatory T cells in HFpEF using global depletion strategies, but to determine whether stress-adaptive signaling within the Treg lineage modifies HFpEF-associated pathology. HFpEF develops in cardiometabolic and hypertensive states where immune dysregulation and reduced Treg abundance have already been reported. STIM1 was therefore selected as a stress-responsive calcium and ER signaling node relevant to immune cell survival and inflammation. Treg-specific STIM1 deletion allows interrogation of cell-intrinsic stress mechanisms without inducing the systemic immune disruption associated with Treg ablation models, which are difficult to interpret in a multifactorial disease such as HFpEF.

Material & methods

Human blood treg isolation and frequency analysis by flow cytometry and qRT-PCR for STIM1

All experimental procedures adhered to the National Institutes of Health Guide for Patient Care and Protection, as well as the Care and Use of Laboratory Animals and institutional guidelines. Approval was granted by the Institutional Review Board (IRB# 21-10-EX-0218-EVMS) and the Animal Care and Use Committees at Macon & Joan Brock Virginia Health Sciences, Old Dominion University (Norfolk, VA), and Wayne State University (Detroit, MI).

Healthy individuals and HFpEF patients’ blood samples were obtained from the Norfolk Sentara General Hospital, VA, USA. 4 mL of blood collected in anti-coagulant tubes was mixed with 4 mL of PBS in a 50 mL tube. Subsequently, in another 50 mL tube containing 8 mL of Ficoll-Histopaque, the blood was slowly added without allowing it to settle, followed by centrifugation at 1400 rpm for 30 min at room temperature. Peripheral blood mononuclear cells (PBMCs) were collected from the interphase of the white buffy coat between the red blood cells (above) and below the plasma. Cell counting was performed using AOPI dye and a cellometer. Cells obtained from the Ficoll gradient were then stained for T-reg cells. A total of 1 × 10⁶ cells were blocked with a Monocyte stain blocker and stained with fluorochrome anti-markers to T-reg cells in Brilliant Stain Buffer Plus (Cat # 566385) from BD Biosciences on ice for 30 min according to the manufacturer’s recommendations. After incubation, cells were washed with MACS buffer at 2000 rpm for 5 min at 4 °C. The supernatant was decanted, and the pellet was resuspended in 200 µl MACS buffer. Stained single-cell suspensions were acquired in Cytekaurora DXP8 color, and unmixing and data analysis were performed using Cytek software. Antibodies used (Supplementary Table 1) were obtained from Biolegend: True Stain Monocyte Blocker (Cat # 426103) and Zombie NIR (Cat # 423106) for live and dead cell separation were purchased from BD Bioscience. Further, Total RNA was isolated from PBMCs T-reg collected from the Ficoll gradient using the MagMAX RNA isolation kit (Ref: AM1830, Lot 01257092, Thermo Fisher Scientific). Subsequently, cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit (Iscript Bio-Rad #1708840), following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was conducted using TaqMan Fast Advanced Master mix (CAT#: 4444556, Thermo Scientific) with specific probes for amplification of Stim1 (NM_003156, CAT#: HP206733), Caspase 3 (NM_004346, CAT#: HP207674), COX2 (PTGS2) (NM_000963, CAT#: HP200900), CHOP (DDIT3) (NM_004083, Cat#: HP207450), PERK (EIF2AK3) (NM_004836, Cat#: HP208096) and GAPDH Mouse qPCR Primer Pair (NM_002046, CAT#: HP205798) for normalization. The relative mRNA expression was calculated using the 2-ΔΔ CT method.

Mice

All the experimental procedures are conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee at the Eastern Virginia Medical School approved them. We bred STIM1^flx/flx mice (Strain #:023350, B6.Cg-STIM1^tm1Rao/J, Jackson Laboratory, USA. These mice carry loxP sites around exon 2 of the STIM1 gene, allowing for conditional mutations in studies related to store-operated Ca2 + entry and Ca2 + homeostasis regulation, with Cre-mice for Foxp3 (Strain #:016959, B6.129(Cg)-Foxp3^{tm4(YFP/icre)Ayr/J}, Jackson Laboratory, USA). Foxp3YFP/cre mice feature YFP and iCre downstream of the Foxp3 gene, enabling the study of regulatory T (T reg) cell development. YFP expression is observable in FOXP3 + Treg cells, and breeding with loxP-flanked sequence mice leads to Cre-mediated recombination in Treg cells, causing deletion of the floxed sequences in the offspring, to generate mice lacking STIM1, specifically in Treg cells. Eight-week-old male Stim1^flox/flox and Treg^Stim1−/− were housed in a temperature-controlled room (22 ± 1˚C), exposed to a 12-hour dark-light cycle, and given access to water and food.

HFpEF induction

Group 1: Stim1^flox/flox and Group 3: Treg^Stim1−/− male mice were fed a regular chow research diet and considered as control groups. Research Diet for the high-fat diet groups (HFD Rodent Diet With 60 kcal% Fat, Research Diets Inc. Catalog# D12492i) with N[w]-nitro-l-arginine methyl ester (L-NAME, 50 mg/100 ml, pH 7.4, CAS number: 51298-62-5, Sigma-Aldrich), Group 2: Stim1^flox/flox + HFD + L-NAME, Group 4: Treg^Stim1−/− + HFD + L-NAME (n = 5–12), was supplied in the drinking water for five weeks are considered the HFpEF mice. We changed the L-NAME solution every other day. At the end of the treatment, mice were euthanized using isoflurane (5%) overdose, followed by heart excision when fully sedated.

Extended material and methods: please see supplementary material & methods

Results

Patient Demographics: The mean age of the HFpEF cohort was 65.36 ± 6.20 years, with a BMI of 33.29 kg/m2. Comorbidities included 37.5% hypertension and 54.5% obese. Despite these differences, all patients exhibited diastolic dysfunction, as evidenced by a significant increase in the E/E’ ratio, with no change in cardiac ejection fraction (EF) (Fig. 1A). Additionally, other metabolic diseases, including obesity, were present (Table 1 and Supplementary Fig. 1). Moreover, we isolated Treg cells from blood samples obtained from healthy individuals and patients with HFpEF. We performed flow cytometry and qRT-PCR to assess Treg cell numbers and the expression of STIM1, apoptosis, inflammation, and ER stress markers. Flow cytometry analysis revealed a higher proportion of Treg cells in healthy individuals than in HFpEF patients, who exhibited a significant reduction in Treg cell numbers (Fig. 1B). Furthermore, the mRNA expression of STIM1, Caspase-3, COX2, and ER stress markers (CHOP and PERK) was markedly upregulated in Treg cells from patients with HFpEF compared to healthy controls (Fig. 1C). These data suggest an association between STIM1 expression and the levels of apoptosis/inflammation/ER stress in Treg cells, as well as Treg cell frequency, in HFpEF.

Fig. 1.

Clinical Characteristics of Control and HFpEF patients’ blood samples. A Evaluation of cardiac function in control (None HFpEF) and HFpEF patients, including left ventricular ejection fraction (EF) and E/E′ ratio, representing reduced systolic function and elevated diastolic filling pressures in HFpEF patients (n = 4–10) (Ob-HT Obese-Hypertensive, N Ob-NT-Non-Obese-Normotensive). B Representative flow cytometry plots illustrating the gating strategy for identifying CD4⁺CD25⁺Foxp3⁺ Treg cells in peripheral blood, with quantification showing a significant decrease in Treg cell frequency in HFpEF patients compared to controls (None HFpEF) patients. C qRT-PCR analysis of Treg cells isolated from patient blood samples showing mRNA expression levels of STIM1, Caspase-3, COX2, CHOP, and PERK. Treg cells from HFpEF patients exhibit elevated expression of genes associated with apoptosis, inflammation, and endoplasmic reticulum (ER) stress compared with controls (None HFpEF) (n = 4–10). Data are shown as mean ± SEM, Student unpaired t-test applied for (A-C), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for None HFpEF (CTR) vs. HFpEF (n = 4–10)

Table 1.

Clinical and Demographic Characteristics of control HFpEF Patients

To test whether this pathway is mechanistically linked to HFpEF. The use of a Foxp3-Cre-mediated STIM1 deletion was intentional and appropriate for the biological question addressed. While STIM1 plays roles in T cell development, Foxp3-Cre activity is restricted to the regulatory T cell lineage and does not broadly target early thymocyte populations. Importantly, the objective of this study was not to dissociate developmental versus inducible effects of STIM1, but to determine whether loss of STIM1-dependent Treg fitness across the lifespan alters susceptibility to HFpEF-associated pathology under cardiometabolic stress. We generated Treg-specific STIM1 knockout mice (Treg^Stim1−/−) using STIM1^flx/flx (B6.Cg-STIM1^tm1Rao/J) with Cre-mice for Foxp3 (B6.129(Cg)-Foxp3^{tm4(YFP/icre)Ayr/J}) mice. Validation of STIM1 deletion and Treg populations have been previously reported in our published research studies in Redox Biology [26] and Translational Research [27]. These studies provided detailed validation of STIM1 deletion in Treg cells and comprehensive characterization of the Treg population. In this context, constitutive Treg-specific deletion captures the integrated contribution of developmental programming and stress-adaptive signaling, which is highly relevant to a chronic disease such as HFpEF. These Treg^Stim1−/− mice and their control (Stim1^flox/flox) were fed a high-fat diet and L-NAME in the drinking water for 5 weeks to induce HFpEF. We measured Foxp3 levels (a specific marker for Treg cells) in heart tissue from all mouse groups. Data illustrate that Foxp3 level was significantly reduced in Stim1^flox/flox subjected to HFpEF compared to Stim1^flox/flox without HFpEF and Treg^Stim1−/− with and without HFpEF mice (Fig. 2A). These findings suggest altered Treg-associated immune signaling in male Stim1^flox/flox mice with HFpEF, indicating a potential role for Treg cell STIM1 expression in reducing Treg cell number in HFpEF pathology. Accordingly, these findings should be interpreted as evidence of altered Treg-associated immune responses rather than definitive quantification of tissue-resident Treg populations.

Fig. 2.

FOXP3 Expression in Heart Tissue and Mouse Treg Isolation and Combo Treatment: A FOXP3 expression level in heart tissue from Stim1^floxflox and Treg^Stim1−/− mice with and without HFpEF. B-E Stim1, Caspase 3, Caspase 12, and CHOP mRNA levels in isolated Treg cells from the spleens of male Stim1^flox/flox mice stimulated for 4 h with or without Combo (n = 3). One-way ANOVA followed by Tukey’s multiple comparisons post hoc test was applied for A. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for Stim1^flox/flox vs. Stim1^flox/flox HFpEF vs. Treg^Stim1−/−vs. Treg^Stim1−/−HFpEF; (n = 5). Student unpaired t-test applied for B-E, **P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for Treg from Stim1^flox/flox and Treg from Stim1^flox/flox + Combo. We used n = 3 mice per experiment, for a total of n = 3

To determine whether this reduction in Treg cells reflects a direct effect of HFpEF-associated stressors on Treg survival, we next performed mechanistic studies using isolated Treg cells from the spleens of male Stim1^flox/flox mice. We then subjected them to a Combo treatment (high glucose, lipid mixture, palmitic acid, and angiotensin II) to simulate HFpEF conditions. After 4 h, qRT-PCR revealed significant upregulation of STIM1 (Fig. 2B) and increased expression of Caspase 3, Caspase 12, and ER stress CHOP compared to Stim1^flox/flox control Treg cells (Fig. 2C-E). These findings suggest that HFpEF induces apoptosis in Treg cells, thereby activating Caspase-dependent apoptotic pathways. This mechanism suggests that HFpEF-associated stressors may disrupt Treg function by promoting Stim1-driven apoptosis, potentially contributing to a pro-inflammatory and stress state that facilitates disease progression.

Having established that HFpEF-associated stressors induce STIM1-dependent apoptosis in Treg cells and that Treg-specific deletion of STIM1 preserves Treg cell numbers, we next examined whether these immune alterations translate into functional cardiac abnormalities characteristic of HFpEF. Cardiac function was assessed by echocardiography (Vevo 3100). Our data illustrated that stim1^flox/flox male mice fed a high-fat diet and L-NAME in the drinking water for 5 weeks develop HFpEF characterized by diastolic dysfunction (increased E/E’), no change in cardiac ejection fraction (E/F), reduced cardiac index, and increased myocardial performance (MPI) (Fig. 3A-D). We didn’t observe a significant change in the E/A ratio or heart rate (Fig. 3E, F).

Fig. 3.

‘Two-hit’ mouse model of HFpEF. Mice fed an HFD + L-NAME for 5 weeks display the key alterations in clinical HFpEF. Echocardiography shows A cardiac ejection fraction (EF), B E/E’ ratio, C cardiac index (CI), D myocardial performance index (MPI), E E/A ratio, and (F) heart rate (bpm) in Stim1^flox/flox, Stim1^flox/flox HFpEF, Treg^Stim1−/− and Treg^Stim1−/− HFpEF mice. One-way ANOVA followed by Tukey’s multiple comparisons post hoc test was applied for A-F. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for Stim1^flox/flox vs. Stim1^flox/flox HFpEF vs. Treg^Stim1−/−vs. Treg^Stim1−/−HFpEF; (n = 3–5). G, H Body weight, I heart weight/tibia length, J lung wet weight/tibia length, K systolic blood pressure (SBP), L reduced running distance, M glucose tolerance test, and N GTT-AUC in Stim1^flox/flox, Stim1^flox/flox HFpEF, Treg^Stim1−/− and Treg^Stim1−/− HFpEF mice. RM-two-way ANOVA applied for H, K, L M, N and One-way ANOVA followed by Tukey’s multiple comparisons post hoc test applied for G, I, J. ns: not significant, RM: Repeated Measurement, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for Stim1^flox/flox vs. Stim1^flox/flox HFpEF vs. Treg^Stim1−/−vs. Treg^Stim1−/−HFpEF; (n = 5–11)

Moreover, stim1^flox/flox male mice with HFpEF display an increase in body weight, heart and lung wet weight, systolic blood pressure (SBP), low exercise performance, and glucose metabolism dysregulation compared to stim1^flox/flox without HFpEF and Treg^Stim1−/− male mice with and without HFpEF (Fig. 3G-N). Interestingly, our data uncovered that Treg^Stim1−/− male mice are protected from HFpEF pathogenesis induced by a high-fat diet and L-NAME in the drinking water for 5 weeks (Fig. 3G-N). Thus, after 5 weeks of a high-fat diet and L-NAME, Treg^Stim1−/− male mice did not develop hypertension and type 2 diabetes/obesity. Instead, they displayed improved cardiac functions, exercise performance, and glucose metabolism (Fig. 3G-N). These data highlight that loss of Treg STIM1 attenuates HFpEF-associated cardiac and systemic pathology.

It is well known that HFpEF is associated with vascular endothelial dysfunction. We assessed vascular endothelial function in mesenteric resistance arteries and the Thoracic Aorta in all groups of mice. Our data showed endothelial dysfunction of large and resistance arteries (aorta and mesenteric resistance arteries) in Stim1^flox/flox male mice with HFpEF compared to Stim1^flox/flox without HFpEF and Treg^Stim1−/− male mice with and without HFpEF (Fig. 4A-B). Interestingly, the endothelial function was not impaired in large and resistance arteries from Treg^Stim1−/− male mice fed a high-fat diet and L-NAME in the drinking water for 5 weeks (Fig. 4A-B). Nitric oxide donor-induced endothelium-independent relaxation and phenylephrine-induced contraction were normal in all groups (Fig. 4A-B). These data suggest that HFpEF primarily impairs endothelial function, rather than the vasculature’s smooth muscle response to nitric oxide. Additionally, the findings highlight an interaction between Treg cells and vascular endothelial function in HFpEF, mediated by STIM1 expression in Treg cells. Thus, western blot analysis and Foxp3 ELISA kit assays show a decrease in eNOS phosphorylation (Fig. 4C) and Foxp3 level (Fig. 4D) in MRA tissue from male Stim1^flox/flox mice with HFpEF compared to male Stim1^flox/flox and Treg^Stim1−/− mice without HFpEF (Fig. 4C, D). Interestingly, eNOS phosphorylation and Foxp3 were unaffected in Treg^Stim1−/− male mice fed a high-fat diet and L-NAME (Fig. 4C, D). It is well-established that hypertension and metabolic diseases are associated with inflammation and ER Stress induction. Our qRT-PCR (Fig. 4E) and western blot (Fig. 4F) data showed that mesenteric resistance arteries from Stim1^flox/flox with HFpEF display a significant increase in inflammation markers (COX2, iNOS, IL-1β and NLRP3), and also apoptotic and cleaved caspase 3 (CC3), cleaved caspase 12 (CC12), and ER stress markers (CHOP) compared to Treg^Stim1−/− mice data when both were subjected to an HFD and L-NAME (Fig. 4G).

Fig. 4.

Mesenteric resistance arteries (MRA) and aorta reactivity showing (A-B) contractility in response to sympathetic stimulation (Phenylephrine, PE), endothelium-dependent and independent relaxation in response to acetylcholine (ACh) and sodium nitroprusside (SNP) in Stim1^flox/flox, Stim1^flox/flox HFpEF, Treg^Stim1−/− and Treg^Stim1−/− HFpEF (n = 5). C Western blot analysis and cumulative data for phospho-endothelial nitric-oxide synthase (P-eNOS) in MRA isolated from Stim1^flox/flox, Stim1^flox/flox HFpEF, Treg^Stim1−/− and Treg^Stim1−/− HFpEF mice (n = 5). D ELISA FOXP3 expression level in MRA tissue, E qRT-PCR data in MRA for inflammation (Cox2, iNOS, IL-1β and NLRP3) and F Western blot analysis and cumulative data for inflammatory markers (COX2, iNOS, and NLRP3) and G Western blot analysis and cumulative data for Apoptotic and ER stress markers (Cleaved Caspase 3 (CC3), Cleaved Caspase 12 (CC12), and CHOP) in MRA tissue. H Representative image of picrosirius red (PSR) of fibrosis in Aorta tissue, from Stim1^flox/flox, Stim1^flox/flox HFpEF, Treg^Stim1−/− and Treg^Stim1−/− HFpEF mice. One-way ANOVA followed by Tukey’s comparisons post hoc test applied for A-H. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for Stim1^flox/flox vs. Stim1^flox/flox HFpEF vs. Treg^Stim1−/− vs. Treg^Stim1−/−HFpEF; (n = 5)

Collagen accumulation in myocardium and other micro- and macrovascular arteries is another hallmark of HFpEF pathogenesis, contributing to tissue fibrosis and stiffness. In this study, we evaluated thoracic aortic fibrosis using picrosirius red staining (PSR) in tissue samples from all experimental groups. Our analysis revealed a significant increase in collagen deposition in Stim1^flox/flox mice fed a high-fat diet (HFD) and treated with L-NAME, compared with other groups (Fig. 4H). This increased fibrosis was associated with elevated markers of cellular stress and inflammation (Fig. 4H), suggesting a potential mechanistic link between inflammatory processes and fibrotic tissue remodeling in HFpEF. Further, we performed Picrosirius Red (PSR) staining on heart tissue to assess fibrosis (Fig. 5A-B). We measured TGF-β (Fig. 5C), BNP (Fig. 5D), CRP (Fig. 5E), TNF-α (Fig. 5F), and IL-1β (Fig. 5G) levels in plasma using ELISA in all groups of mice. Furthermore, qRT-PCR (Fig. 5H-K) and western blot analysis (Fig. 5L) were conducted to determine the expression levels of Inflammatory markers (COX2, iNOS, IL-1β, and NLRP3), apoptotic and ER stress markers (Cleaved Caspase 3 (CC3), Cleaved Caspase 12 (CC12), and CHOP) in heart tissue (Fig. 5M). Our results demonstrated a significant increase in all these markers in Stim1^flox/flox mice subjected to HFD and L-NAME treatment compared to Stim1^flox/flox control mice and Treg^Stim1−/− mice fed with and without high-fat diet and L-NAME (Fig. 5A-M).

Fig. 5.

Histological and functional analyses of heart tissue and plasma in mice with and without HFpEF. A-B Representative images of Picrosirius Red (PSR) staining illustrating cardiac fibrosis, along with quantitative analysis of fibrotic area, in Stim1^flox/flox, Stim1^flox/flox HFpEF, Treg^Stim1−/− and Treg^Stim1−/− HFpEF mice. Plasma concentrations of fibrotic and inflammatory mediators, including TGF-β (C), BNP (D), CRP (E), TNF-α (F), and IL-1β (G), measured to assess systemic inflammation and cardiac stress across experimental groups. H-K qRT-PCR analysis of Heart tissue demonstrating transcriptional regulation of inflammatory genes (Cox2, iNOS, IL-1β, and NLRP3), L Western blot analysis of Heart tissue lysates with densitometric quantification showing expression of inflammatory proteins (COX2, iNOS and NLRP3) and M Western blot analysis and densitometric quantification showing expression of Apoptotic and ER stress markers (Cleaved Caspase 3 (CC3), Cleaved Caspase 12 (CC12), and CHOP ) in Heart tissue from Stim1^flox/flox, Stim1^flox/flox HFpEF, Treg^Stim1−/− and Treg^Stim1−/− HFpEF mice. One-way ANOVA followed by Tukey’s multiple comparisons post hoc test applied for (A-M). ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for Stim1^flox/flox vs. Stim1^flox/flox HFpEF vs. Treg^Stim1−/−vs. Treg^Stim1−/−HFpEF; (n = 5)

HFpEF is a complex condition that contributes to systemic organ damage. Kidney and brain injury are critical due to the interconnected nature of cardiovascular, renal, and neurological systems. In HFpEF, chronic hypoperfusion, inflammation, and kidney fibrosis are exacerbated by metabolic stressors, such as high-fat diets (HFD), and by nitric oxide inhibition with L-NAME, resulting in elevated markers of tubular damage, including Kidney Injury Molecule-1 (KIM-1) and Lipocalin-2. Similarly, brain injury in HFpEF is associated with neuroinflammation, oxidative stress, and fibrosis, exacerbated by HFD and L-NAME. To investigate these mechanisms, we performed PSR staining to assess kidney fibrosis (Fig. 6A-C) and brain fibrosis (Fig. 6F-G) and measured plasma levels of Kim1 (Fig. 6D) and Lipocalin-2 (Fig. 6E) using ELISA. Our findings showed significant increases in these markers in Stim1^flox/flox mice fed a HFD and L-NAME treatment compared to Stim1^flox/flox control mice and Treg^Stim1−/− mice fed with and without a high-fat diet and L-NAME (Fig. 6A-G).

Fig. 6.

Histological and functional analyses of kidney and brain tissue in mice with and without HFpEF. A-B Representative images of Picrosirius Red (PSR) staining illustrating fibrosis in kidney tissue, along with quantitative analysis of fibrotic area. C Kidney weight measurements as an indicator of organ remodelling and hypertrophy in response to HFpEF, D Plasma concentrations of kidney injury marker-1 (TIM-1/KIM-1/HAVCR) reflecting renal tubular injury. E Plasma Lipocalin-2 levels, serving as an additional marker of renal damage and inflammation level in Stim1^flox/flox, Stim1^flox/flox HFpEF, Treg^Stim1−/− and Treg^Stim1−/− HFpEF mice. F–G Representative images of Picrosirius Red (PSR) staining illustrating fibrosis in brain tissue with corresponding quantitative analysis in Stim1^flox/flox, Stim1^flox/flox HFpEF, Treg^Stim1−/− and Treg^Stim1−/− HFpEF mice. One-way ANOVA followed by Tukey’s multiple comparisons post hoc test applied for (A-G). ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for Stim1^flox/flox vs. Stim1^flox/flox HFpEF vs. Treg^Stim1−/−vs. Treg^Stim1−/−HFpEF; (n = 5)

These in vitro studies were designed to assess inflammatory signaling capacity rather than species-specific cardiomyocyte physiology. Although H9C2 cardiomyoblasts provide a controlled and widely used model for studying conserved stress and inflammatory signaling pathways, these experiments do not fully recapitulate adult cardiomyocyte biology. Accordingly, the mechanistic conclusions derived from H9C2 based assays should be interpreted as supportive and hypothesis-generating evidence rather than definitive evidence of direct cardiomyocyte-specific mechanisms. To investigate the interaction between Treg cells and HFpEF development, we conducted in vitro studies. H9C2 cells (H9C2 - CRL-1446, ATCC) were aseptically cultured in 6-well plates. Treg cells were isolated from stim1^flox/flox and Treg^Stim1−/− mice and stimulated with the combo for 4 h to mimic HFpEF. Control (CTL) H9C2 cells were divided into eight different conditions: (1) H9C2 CTL, (2) H9C2 CTL+ Combo, (3) H9C2 CTL+ Treg from CTL stim1^flox/flox and Treg^Stim1−/−,(4) H9C2 CTL+ Treg from stim1^flox/flox and Treg^Stim1−/− + Combo treated, (5) H9C2 CTL+ recombinant Interleukin-17 A (IL-17R), (6) H9C2 CTL+ Treg from stim1^flox/flox and Treg^Stim1−/− + Combo treated + IL-17 Neutralizing antibody (IL-17 NA_b), (7) H9C2 CTL+ recombinant mouse Interferon gamma (IFNgR), (8) H9C2 CTL+ Treg from stim1^flox/flox and Treg^Stim1−/− + Combo treated + IFNG Neutralizing antibody (IFNg NA_b). After 4 h of T-reg cell incubation, the cells were centrifuged, and the control and combo Treg cells from stim1^flox/flox and Treg^Stim1−/− mice were transferred to Control H9C2 cells for overnight incubation. The next day, the H9C2 cells were harvested, lysed, and analyzed by Western blotting for the inflammatory markers iNOS, COX-2, and NLRP3. The results showed that H9C2 control (CTL) cells treated with the Combo and combo-treated Treg cells from stim1^flox/flox mice, IL-17R, and IFNγR exhibited increased inflammation. However, this inflammation was reduced when H9C2 cells were treated with combination-treated Treg cells from stim1^flox/flox mice in the presence of an IL-17 neutralizing antibody (NA_b) and an IFNγ neutralizing antibody (NA_b) (Fig. 7A). Similarly, H9C2 CTL cells treated with the combo, IL-17R, and IFNγR showed increased inflammatory markers. In contrast, H9C2 CTL cells treated with combo-treated Treg cells from TregStim1-/- mice did not exhibit this increase in inflammation. Furthermore, treatment with combo-treated Treg cells from Treg^Stim1−/− mice and IL-17 NA_b and IFNγ NA_b also reduced inflammation (Fig. 7B). These findings suggest that STIM1 in T-reg cells facilitates IL-17 release and that IFNγ promotes cardiac inflammation. Equally, disruption of STIM1 in Treg cells diminishes this inflammatory response. This suggests that STIM1 contributes to inflammation and the pathogenesis of HFpEF.

Fig. 7.

Co-culture between H9C2 cells and Treg cells from Stim1^flox/flox, Treg^Stim1−/− mice and treated with and without Combo, IL-17, INFg (R), IL-17NAb, and INFg NAb. A-B Western blot analysis and cumulative data for inflammatory factors (iNOS, COX2, and NLRP3) in H9C2 cells (1) H9C2 CTL, (2) H9C2 CTL+ Combo, (3) H9C2 CTL+ Treg from CTL stim1^flox/flox and Treg^Stim1−/−, (4) H9C2 CTL+Treg from stim1^flox/flox and Treg^Stim1−/− + Combo treated, (5) H9C2 CTL+ recombinant Interleukin-17 A (IL-17R), (6) H9C2 CTL+ Treg from stim1^flox/flox and Treg^Stim1−/− + Combo treated + IL-17 Neutralizing antibody (IL-17NA_b), (7) H9C2 CTL+ recombinant mouse Interferon gamma (IFNgR), (8) H9C2 CTL+ Treg from stim1^flox/flox and Treg^Stim1−/− + Combo treated + IFNG Neutralizing antibody (IFNgNA_b). One-way ANOVA followed by Tukey’s multiple comparisons post hoc test applied for (A-B). ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for H9C2 CTL vs. H9C2 CTL+ Combo vs. H9C2 CTL+ Treg from CTL stim1^flox/flox and Treg^Stim1−/− vs. H9C2 CTL+ Treg from stim1^flox/flox and Treg^Stim1−/− + Combo treated vs. H9C2 CTL+ recombinant Interleukin-17 A (IL-17 A vs. H9C2 CTL+ Treg from stim1^flox/flox and Treg^Stim1−/− + Combo treated + IL-17 Neutralizing vs. H9C2 CTL+ recombinant mouse Interferon vs. H9C2 CTL+ Treg from stim1^flox/flox and Treg^Stim1−/− + Combo treated + INFγ Neutralizing antibody (n = 3). To achieve n = 3, we used n = 4 mice for each condition

Discussion

Our novel and significant findings indicate that STIM1 deletion in Treg cells protects against HFpEF pathogenesis, likely through IL-17- and IFN-γ-mediated mechanisms.

Heart failure with preserved ejection fraction (HFpEF) is a multifactorial disease characterized by dysregulation of cardiac diastolic relaxation and vascular dysfunction. It is a significant unmet need in cardiovascular medicine and remains with very limited therapeutic options. HFpEF prevalence is increasing and has become the most common form of heart failure, with severe complications of the cardiovascular system. Understanding the pathogenesis of HFpEF provides us with essential insights into the pathways and mechanisms underlying the disease. Inflammation is widely thought to play a key role in HFpEF pathogenesis and has been considered a significant contributor to cardiovascular diseases for a long time. Patients with HFpEF exhibit signs of chronic inflammation, and recent reports have also shown elevated circulating inflammatory biomarkers, including iNOS, COX2, NLRP3, Interleukin-1b (IL-1β), and CRP [28–33]., In other animal models of HFpEF generated by a long-term high-fat diet and desoxycorticosterone pivalate or high salt diet for 28 weeks [32], the authors showed an increase in inflammatory biomarkers associated with an exacerbation of mitochondrial hyperacetylation. Furthermore, it is well established that systemic inflammation is a significant contributor to the development of cardiovascular disease, including HFpEF [30, 34, 35]. The HFpEF model used here integrates metabolic and nitric oxide-dependent stressors to reflect the multifactorial nature of the disease, and the present study was not designed to isolate the effects of individual disease components but to identify immune pathways that modify disease severity in an integrated HFpEF model.

The immune system’s involvement in Hypertension-induced cardiovascular complications has long been appreciated. For instance, it has been reported that the Treg cells play an essential role in vascular dysfunction in mice infused with angiotensin II [24]. Hypertension is associated with reduced Treg cell numbers, which are linked to vascular dysfunction. The transfer of healthy Treg cells from control mice into hypertensive mice protects the vascular function [12, 13, 24]. Recent studies have suggested a potential association between Treg cells and pulmonary hypertension. Th-17 cells and their associated cytokines are involved in the development of hypertension-mediated organ damage, and reduced numbers of Treg cells are observed in hypertension [36]. Another study reported that transferring healthy Treg cells into hypertensive mice protected the heart from damage independent of blood pressure [37]. Together, these studies highlight the significant role of the immune cells, particularly the immune Treg cells, in hypertension-induced cardiovascular disease. Our study revealed that STIM1 expression was significantly increased in Treg cells isolated from HFpEF patient blood samples and in Tregs from Stim1^flox/flox mice treated with combination (Combo) therapy, compared to non-HFpEF patient samples and control Tregs from Stim1^flox/flox mice. This increase in STIM1 expression was associated with elevated Caspase-3 and Caspase-12. Additionally, the Treg population was markedly reduced in HFpEF patients compared to non-HFpEF samples. Furthermore, Foxp3 expression levels were reduced in Stim1 ^flox/flox mice treated with HFpEF compared with those in control mice and in Tregs lacking STIM1. Importantly, Foxp3 and other tissue level molecular readouts used in this study should be interpreted as indicative of Treg-associated immune modulation rather than definitive immune cell characterization, and do not substitute for comprehensive immune phenotyping approaches such as flow cytometry or functional Treg assays. However, these findings should not be interpreted as a complete or definitive characterization of immune cells based solely on tissue-level markers. These findings are consistent with the possibility that increased STIM1 signaling contributes to Treg stress-associated dysfunction and apoptosis during HFpEF. Thus, targeting STIM1 in Tregs may be a therapeutic strategy to prevent or mitigate HFpEF-related cardiovascular complications. Data from other labs indicate that STIM1 plays distinct roles in endothelial cells, smooth muscle cells (SMCs), and cardiomyocytes. Thus, conditional deletion of STIM1 in endothelial cells causes endothelial dysfunction, while deletion of STIM1 in SMCs does not affect endothelial functions [20]. Moreover, the deletion or overexpression of STIM1 in cardiomyocytes leads to heart failure [22, 23]. These studies highlight that targeting STIM1 for therapy should be cell-type specific. Studies from other laboratories report that STIM1 is expressed in the heart and upregulated under disease conditions. Additionally, transgenic mice with cardiac-specific STIM1 overexpression, mimicking the disease-associated increase in this protein, demonstrate that elevated STIM1 levels contribute to cardiac hypertrophy [38]. Clinical trials reported that an inflammatory blockade in obese patients with HFpEF reduced N-terminal pro-B-type natriuretic peptide levels and improved exercise training [39, 40]. However, Mineralocorticoid receptor blockade enhances coronary microvascular function in type 2 diabetes, while angiotensin II type 1 receptor blockade exerts anti-inflammatory effects in hypertensive patients with microinflammation [41, 42]. However, recent clinical studies with anti-inflammatory agents have shown negative outcomes in patients with HFpEF [39, 40]. In the present study, we aimed to determine the significant impact of STIM1 in HFpEF pathogenesis by deleting the STIM1 gene in Treg cells. Our findings reveal a critical role for STIM1 in the pathogenesis of HFpEF. Specifically, the mice with Treg cell-specific deletion of the Stim1 gene were protected from HFpEF development induced by a high-fat diet combined with L-NAME administration for five weeks. Given the established role of STIM1 in Th17 differentiation, it is plausible that STIM1 deletion in Treg cells limits Treg instability or conversion toward pro-inflammatory Th17-like phenotypes under metabolic stress, a possibility that warrants future investigation.

Most patients with HFpEF harbor the morbidity of hypertension, obesity/type 2 diabetes/metabolic syndrome [7, 8]. Our preclinical mouse model of HFpEF recapitulates most clinical features of the disease in patients [14]. Using a high-fat diet and L-NAME in the drinking water for five weeks, the stim1^flox/flox male mice exhibited not only cardiac abnormalities (diastolic dysfunction, hypertrophy, inflammation, and fibrosis) but also hypertension, metabolic disorders, increased body weights, heart weight, kidney, lung weight, impaired exercise tolerance, increased blood glucose level, kidney injury, while Treg^Stim1−/− mice fed the same high-fat diet, and L-NAME significantly improved these parameters.

The protection of micro- and macrovascular endothelial function may result from multiple factors, including reduced inflammation in the aorta and mesenteric resistance arteries. It has been previously shown that rats treated with L-NAME for three weeks exhibited reduced flow-induced dilation in mesenteric arteries, which was restored by quinapril [43]. Moreover, as previously reported, in mesenteric resistance arteries, there is the possibility of a switch in the vasodilators involved in endothelium-dependent relaxation, favoring prostaglandins [43].

Our data showed endothelial dysfunction of large and resistance arteries in Stim1^flox/flox male mice fed a high-fat diet and L-NAME compared with Stim1^flox/flox and Treg^Stim1−/− male mice. Remarkably, the endothelial function was not impaired in large and resistance arteries from Treg^Stim1−/− male mice fed a high-fat diet and L-NAME. Nitric oxide donor-induced endothelium-independent relaxation and phenylephrine-induced contraction were normal in groups fed a high-fat diet and L-NAME. These data signify that HFpEF compromises the endothelial function rather than the response of vascular smooth muscle cells to nitric oxide, and deletion of Stim1 in Treg cells blunted the induction of inflammation, cardiac, kidney, brain, and arterial fibrosis and protected the metabolism from dysregulation.

Supporting evidence from other studies underscores the role of STIM1 in cardiac pathology. STIM1 expression is upregulated in the heart under disease conditions, and cardiac-specific overexpression of STIM1 in transgenic mice, mimicking this pathological elevation, drives the development of cardiac hypertrophy [23, 38]. Moreover, adoptive transfer of healthy Treg cells into hypertensive mice has been shown to mitigate cardiac damage independent of blood pressure regulation [37]. Collectively, these findings highlight the pivotal role of immune modulation by Treg cells in mitigating inflammation and underscore their therapeutic potential in HFpEF pathogenesis, with STIM1 serving as a key molecular target in this context.

HFpEF is increasingly recognized as a systemic syndrome characterized by multiorgan dysfunction rather than an isolated cardiac disorder. Emerging clinical and experimental evidence indicates that chronic systemic inflammation, endothelial dysfunction, and microvascular injury contribute to pathological remodeling in extra-cardiac organs, including the kidney and brain [7, 44]. In HFpEF, renal fibrosis and tubular injury are commonly observed and are closely associated with inflammatory cytokine signaling and microvascular impairment [45]. Similarly, neuroinflammation and cerebral microvascular damage have been reported in HFpEF and related cardiometabolic conditions, contributing to cognitive decline and structural brain alterations [46]. Given the interconnected nature of cardiovascular, renal, and neurological systems, metabolic stressors such as a high-fat diet (HFD) and nitric oxide synthase inhibition with L-NAME exacerbate systemic inflammation, hypoperfusion, and fibrotic remodeling. These conditions are associated with elevated markers of renal tubular injury, including Kidney Injury Molecule-1 (KIM-1) and Lipocalin-2, as well as neuroinflammation and brain fibrotic changes. Therefore, we evaluated kidney and brain fibrosis to determine whether Treg-specific STIM1 signaling contributes to HFpEF-associated systemic inflammatory injury. The reciprocal interactions and underlying mechanisms linking inflammation and fibrosis demand detailed study. Our findings demonstrate that Stim1 deletion in Treg cells significantly attenuates inflammation induced by a high-fat diet and L-NAME treatment. Specifically, the absence of Stim1 in Treg cells led to reduced expression of key inflammatory markers, including iNOS, COX2, IL-1β, TNF-alpha, CRP, the inflammasome component NLRP3 and apoptotic and ER stress markers (Caspase3, Caspase 12 and CHOP) Consistently, Treg Stim1^−/− mice subjected to HFpEF conditions exhibited a markedly reduced fibrosis across multiple organs, including the T. Aorta, Heart, Kidney, and Brain. Plasma level of KIM-1, a marker of kidney injury, and Lipocalin-2, a marker of systemic and brain inflammation, was significantly lower in the Treg Stim1^−/− HFpEF mice compared to Stim1^flox/flox HFpEF mice. Plasma circulating Fibrotic markers, including BNP and TGF-β levels, were also reduced, indicating protection against multi-organ fibrotic injury. These results highlight STIM1 as a key mediator of HFpEF progression and suggest its potential as a therapeutic target to prevent and treat HFpEF.

STIM1, a critical regulator of Store-Operated Calcium Entry (SOCE), modulates intracellular calcium signaling in immune cells, thereby impacting inflammation and fibrosis. IL-17, primarily produced by Th17 cells, promotes neutrophil and macrophage recruitment, endothelial dysfunction, and myocardial fibrosis by driving oxidative stress and upregulating mediators such as iNOS, COX2, and NLRP3 inflammasomes. IFN-γ, secreted by Th1 and NK cells, plays a dual role: exacerbating inflammation by activating macrophages and promoting cytokine release (e.g., IL-1β, TNF-α), while mitigating fibrosis by limiting extracellular matrix deposition. In HFpEF, IFN-γ may modulate immune responses to reduce cardiac hypertrophy and fibrosis.

The interaction between STIM1 deletion in Treg cells and in H9C2 cells provides supportive experimental evidence for the development of cardiovascular inflammation, fibrosis, and HFpEF, which remain unknown. However, these mechanistic observations are based on H9C2 cardiomyoblasts and therefore represent supportive experimental evidence. Therefore, these findings should be interpreted with caution and primarily as mechanistic support for inflammatory signaling pathways, rather than as direct evidence of adult cardiomyocyte behavior in HFpEF. Furthermore, our in vitro findings provide direct evidence that H9C2 control (CTL) cells treated with the combo, along with combo-treated Treg cells from stim1^flox/flox mice, IL-17R, and IFNγR, exhibited increased inflammation. However, this inflammation was reduced when H9C2 cells were treated with combo-treated Treg cells from stim1^flox/flox mice in the presence of IL-17 neutralizing antibody (NAb) and IFNγ neutralizing antibody (NAb). Similarly, H9C2 CTL cells treated with the combo, IL-17R, and IFNγR showed an increase in inflammatory markers, whereas H9C2 CTL cells treated with combo-treated Treg cells from Treg^Stim1−/− mice did not. Furthermore, treatment with combo-treated Treg cells from Treg^Stim1−/− mice, and with IL-17 NAb and IFNγ NAb, also reduced inflammation. These findings suggest that STIM1 in T-reg cells facilitates the release of IL-17 and IFNγ, promoting cardiac inflammation. Equally, disruption of STIM1 in Treg cells diminishes this inflammatory response. This suggests that STIM1 contributes to the development of inflammation and fibrosis in the pathogenesis of HFpEF.

Conclusion

This study uncovered a significant and novel mechanism by which STIM1 in Treg cells contributes to HFpEF pathogenesis, possibly via inflammation. The findings suggest that selectively modulating STIM1 in Treg cells could be a promising therapeutic strategy to prevent or treat cardiovascular complications of HFpEF.

Limitations and future directions included in the revised manuscript

This study should be interpreted in the context of several limitations. First, mechanistic in vitro experiments were performed using H9C2 cardiomyoblasts, which, although widely used to study cardiomyocyte stress and inflammatory signaling, do not fully recapitulate the phenotype of adult cardiomyocytes. While these experiments were designed to provide controlled mechanistic insight and are supported by robust in vivo findings, future studies will validate these pathways in more physiologically relevant systems, including primary adult cardiomyocytes and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes.

Second, our genetic strategy relied on floxed littermate controls and did not include Cre-expressing control mice. Although this approach is standard in conditional knockout models and Cre-loxP recombination-based Foxp3-Cre systems have not been associated with significant Cre-driven phenotypes, we acknowledge that the inclusion of Cre-only controls would further strengthen causal interpretation. Future studies will incorporate these controls to definitively exclude potential Cre-related effects.

Third, comprehensive immune profiling of myocardial and peripheral compartments was not performed. Although our study supports functional and molecular evidence supporting Treg-mediated mechanisms, detailed characterization of immune cell populations, including myocardial Tregs, would provide additional mechanistic resolution. In particular, Foxp3-based analyses and related tissue-level molecular markers were used as surrogate indicators of Treg-associated immune responses and should not be interpreted as full or definitive immune cell phenotyping. Specifically, the absence of cardiac flow cytometry analysis, along with the lack of direct ex vivo characterization of splenic and peripheral Treg populations, limits our ability to fully define changes in Treg abundance, phenotype, and functional status across relevant immune compartments during HFpEF.

Future work will integrate advanced immune profiling approaches, such as cardiac flow cytometry and single-cell transcriptomics, to define immune cell dynamics and their contribution to disease progression.

Despite these limitations, the current study provides strong in vivo and mechanistic evidence supporting a critical role for Treg-dependent pathways in cardiovascular pathology. Future investigations addressing these gaps will further refine the cellular and molecular mechanisms and enhance translational relevance.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

BS, Ak, PH, XJ, PF (Experiments, data generation, graphs, writing MS) IM (Statistics) JM (Human data) NR (Supervise XJ, PF - Data- analysis, and writing MS) RB and JH (Human data, analysis, and writing MS) KM (Concept, data analysis, supervise the project, writing MS).

Funding

This work is supported by NIH-HL150014 (KM), NIH-HL151616 (KM), and NIH-HL136456 (NER).

Data availability

Data are available upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.