Catestatin Protects Against Diastolic Dysfunction by Attenuating Mitochondrial Reactive Oxygen Species Generation

Abstract

Background

Catestatin has been reported as a pleiotropic cardioprotective peptide. Heart failure with preserved ejection fraction (HFpEF) was considered a heterogeneous syndrome with a complex cause. We sought to investigate the role of catestatin in HFpEF and diastolic dysfunction.

METHODS AND RESULTS

Administration of recombinant catestatin (1.5 mg/kg/d) improved diastolic dysfunction and left ventricular chamber stiffness in transverse aortic constriction mice with deoxycorticosterone acetate pellet implantation, as reflected by Doppler tissue imaging and pressure‐volume loop catheter. Less cardiac hypertrophy and myocardial fibrosis was observed, and transcriptomic analysis revealed downregulation of mitochondrial electron transport chain components after catestatin treatment. Catestatin reversed mitochondrial structural and respiratory chain component abnormality, decreased mitochondrial proton leak, and reactive oxygen species generation in myocardium. Excessive oxidative stress induced by Ru360 abolished catestatin treatment effects on HFpEF‐like cardiomyocytes in vitro, indicating the beneficial role of catestatin in HFpEF as a mitochondrial ETC modulator. The serum concentration of catestatin was tested among 81 patients with HFpEF and 76 non–heart failure controls. Compared with control subjects, serum catestatin concentration was higher in patients with HFpEF and positively correlated with E velocity to mitral annular e′ velocity ratio, indicating a feedback compensation role of catestatin in HFpEF.

Conclusions

Catestatin protects against diastolic dysfunction in HFpEF through attenuating mitochondrial electron transport chain–derived reactive oxygen species generation. Serum catestatin concentration is elevated in patients with HFpEF, probably as a relatively insufficient but self‐compensatory mechanism.

Nonstandard Abbreviations and Acronyms

CATSTAT‐HF

Serum Catestatin Expression and Cardiometabolic Parameters in Patients With Congestive Heart Failure

DOCA

deoxycorticosterone acetate

E/e′

E velocity to mitral annular e′ velocity ratio

ETC

electron transport chain

HFpEF

heart failure with preserved ejection fraction

ROS

reactive oxygen species

TAC

transverse aortic constriction

Clinical Perspective.

What Is New?

• Exogenous catestatin supplementation mitigates diastolic function and cardiac remodeling in a transverse aortic constriction/deoxycorticosterone acetate–induced heart failure with preserved ejection fraction (HFpEF) mice model.

• Catestatin restored cardiomyocyte mitochondrial electron transport chain abnormality and reduced the reactive oxygen species generation in HFpEF.

• Serum catestatin concentration is elevated in patients with HFpEF, as a relatively insufficient but self‐compensatory mechanism.

What Are the Clinical Implications?

• As an endogenous negative feedback hormone, catestatin supplementation can protect against diastolic dysfunction in HFpEF.

• Recognizing altered mitochondrial respiratory chain and oxidative stress homeostasis in HFpEF may provide new therapeutic options for treating HFpEF.

Heart failure (HF) with preserved ejection fraction (HFpEF) represents >50% of all HF, and its prevalence continues to increase owing to the overweight, aging, and chronically diseased population. ¹ Considered a heterogeneous disorder, the pathophysiology underpinning HFpEF remains relatively unclear. HFpEF has become a major cause of morbidity and mortality with scarce effective therapy.

Catestatin is a bioactive proteolytical 21‐amino‐acid‐residue from the chromogranin A that inhibits catecholamine secretion by antagonizing nicotine acetylcholine receptors. ² Diverse cardioprotective effects of catestatin have been recognized, including antihypertension, ³ inotropy regulation, ⁴ calcium flux modulation, ⁵ etc. We also have recently reported the antiatherogenic effects of catestatin by modulating the endothelium‐leukocyte interaction via an angiotensin‐converting enzyme 2–dependent mechanism. ⁶ Although neuroendocrine overactivation, inflammation, and prolonged calcium transient all contributed to HFpEF pathogenesis, the therapeutic role of catestatin in HFpEF remains indeterminate.

Diastolic dysfunction associated with impaired left ventricular (LV) relaxation and myocardial stiffening is the fundamental pathological change of HFpEF. ⁷ A role for elevated myocardial reactive oxygen species (ROS) in diastolic dysfunction development and severity was previously reported. ⁸ Mitochondria is the major site for free radical generation. ⁹ Mitochondria abnormality in HFpEF disrupts the electron transport chain (ETC), leading to extensive electron leakage and subsequent oxidant formation ¹⁰ that mainly originated from complex I and complex III. Moreover, complex I inhibition and ROS scavenger protects cardiomyocytes from oxidative damage and improves cell survival. ¹¹ , ¹² However, a potential translational approach to regulate oxidative stress in HFpEF is unclear.

Here, we investigated the role of catestatin in diastolic dysfunction. Transverse aortic constriction (TAC) mice with deoxycorticosterone acetate (DOCA) pellet implantation were used to study the therapeutic approach to diastolic dysfunction in HFpEF. We revealed that exogenous catestatin supplementation protects against diastolic dysfunction by attenuating mitochondria‐derived ROS generation. Moreover, serum catestatin concentration and its clinical relevance was tested in a HFpEF cohort.

Method

The data that support the findings of this study are available from the corresponding author upon reasonable request. More detailed methods are provided in Data S1.

Animals and Experiment Protocol

All animal experiments were carried out following the Guide for the Care and Use of Laboratory Animals, approved by the Animal Care and Use Committee of Shanghai Jiao Tong University (China), and followed the US National Institutes of Health Using Animals in Intramural Research guidelines for animal use. All mice we used were male C57BL/6 mice aged 8 to 10 weeks and were rested at least 5 days before randomization.

TAC was performed as described previously. ¹³ The transverse aortic arch was gently exposed and ligated by tying a 5–0 silk suture ligature against a 27‐gauge needle. A 50‐mg DOCA pellet was implanted subcutaneously during TAC surgery and was released in 21 days. Sham animals underwent the thoracotomy without the aortic ligation, and a placebo pellet was implanted. Three weeks after surgery, a follow‐up echocardiographic study was performed to evaluate heart function. The TAC/DOCA group and sham group were randomly divided into 2 groups (n=8 per group), respectively. One was injected intraperitoneally with recombinant human catestatin (CgA352‐372, SSMKLSFRARGYGFRGPGPQL, dissolved in PBS, 1.5 mg/kg body weight) and the other with PBS only (same volume as catestatin group) for 28 consecutive days. After 4 weeks of injection, a final echocardiography was performed, and then pressure‐volume analysis, isolated cardiomyocyte studies, RNA sequencing, and protein analysis were carried out.

Human HFpEF Cohort

Blood samples and deidentified individual clinical data of 81 patients with HFpEF and 76 participants without HF were obtained from the Risk Evaluation and Management in Heart Failure Trial (NCT02998788) database. ¹⁴ The research was approved by the Institutional Review Board of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. All participants provided written informed consent forms. Subjects without HF were healthy controls with echocardiographic confirmation of the absence of HF. Subjects with HFpEF were de novo clinically diagnosed with HF with an LV ejection fraction ≥50%.

Blood samples were obtained from all participants after at least 8 hours of overnight fasting and 2 hours of rest in the supine position and were analyzed in the central laboratory of Ruijin Hospital. All blood samples were transferred at freezing temperature and centrifuged within 20 minutes at 1200 g to obtain serum. All samples were stored at −80°C before analysis. Serum catestatin concentration was measured with a commercial human catestatin ELISA kit (Cat# EK‐053‐27, Phoenix Pharmaceuticals, Burlingame, CA) according to the product instructions.

Statistical Analysis

Data were expressed as mean±SD or median (interquartile range), as appropriate. Prism 9.0 software (GraphPad Software Inc, La Jolla, CA) and R software version 4.2.0 (R Foundation for Statistical Computing, Vienna, Austria) were used for the statistical analysis. Normality of the data was assessed by the Shapiro–Wilk test. Differences between the 2 groups were compared using 2‐tailed unpaired Student t tests when normal distribution was satisfied; otherwise, the Mann–Whitney nonparametric test was used. Differences among ≥3 groups were compared using 1‐way ANOVA followed by the Tukey post hoc test when the assumption was satisfied (normal distribution). Otherwise, the nonparametric Kruskal–Wallis test followed by Dunn's multiple comparisons test was used for further analysis. The correlation between catestatin and other clinical parameters was analyzed by Spearman rank correlation analysis. P<0.05 was considered to be statistically significant.

Results

Catestatin Protects Diastolic Dysfunction in Mice With HFpEF

To explore the effect of catestatin in HFpEF, diastolic dysfunction was induced in 2‐month‐old male C57BL/6 mice using a well‐established TAC/DOCA model that accelerates cardiac remodeling and LV stiffening through chronic pressure overload and mineralocorticoid excess–associated oxidative stress. ¹⁵ At 3 weeks after surgery, all TAC/DOCA mice developed diastolic dysfunction with systolic function preserved, while intact LV function was retained in the sham group (Table S1). Animals were then subjected to intraperitoneal injection with either vehicle or catestatin at a concentration of 1.5 mg/kg body weight per day. These mice are referred to as sham, sham+catestatin, TAC/DOCA, and TAC/DOCA+catestatin, respectively. After 4 weeks of consecutive catestatin intervention, the animals were studied (Figure 1A).

Figure 1. CST protects diastolic dysfunction in mice with heart failure with preserved ejection fraction.

A, Schematic of animal experimental protocol. Three weeks after TAC with DOCA pellet implantation or sham surgery, mice were subjected to vehicle or catestatin (1.5 mg/kg/d) administration for 4 weeks. These mice are referred to as sham, sham+catestatin, TAC/DOCA, and TAC/DOCA+catestatin, respectively. B, Echocardiographic analysis of LVEF at week 7. C, Representative images of mitral annular Doppler tissue imaging at week 7. D through F, Doppler analysis of mitral E/A ratio (D), mitral E wave deceleration time (E), and mitral E/e′ ratio (F) were measured at week 7 (n=8 per group). G and H, Representative steady‐state left ventricular PV loops. I through L, The diastolic stiffness coefficient β of EDPVR (I), LVESP (J), dp/dt (K) and −dp/dt (L) were calculated from the PV loop analysis (n=5 per group). Data are presented as mean ± SD and analyzed using 1‐way ANOVA followed by Tukey multiple comparisons test. **P<0.01, ***P<0.001 vs sham group; ^# P<0.05, ^## P<0.01, ^### P<0.001 vs TAC/DOCA group. CST indicates catestatin; DOCA, deoxycorticosterone acetate; dp/dt, systolic contractility; ‐dp/dt, diastolic relaxation; EDPVR, end‐diastolic PV relationship; LVEF, left ventricular ejection fraction; LVESP, left ventricular end‐systolic pressure; PV, pressure volume; and TAC, transverse aortic constriction.

At the end of the 7‐week period, the systolic function remained preserved among all 4 groups (Figure 1B). Though a slight reduction in LV ejection fraction was detected in the TAC/DOCA group, it is still >50%. Representative results of mitral annular velocity by Doppler tissue imaging are shown in Figure 1C. An increase in transmitral E/A velocity ratio (Figure 1D) and reduction in E wave deceleration time (Figure 1E) was still observed in TAC/DOCA mice, suggesting a restrictive LV diastolic filling pattern and LV stiffening. The E velocity to mitral annular e′ velocity ratio (E/e′), a universal predictor of LV end‐diastolic filling pressure, was also elevated dramatically in the TAC/DOCA group (Figure 1F). Importantly, all these parameters reflecting diastolic dysfunction were partly reversed in the TAC/DOCA+catestatin group, suggesting a therapeutic role of catestatin in HFpEF. Moreover, no adverse additive effects were observed in the sham+catestatin mice.

Pressure‐volume analysis via a conductance catheter was also performed at the 7‐week postsurgery time point with representative loops shown in Figure 1G and 1H. To quantify LV chamber stiffness, the load‐independent diastolic stiffness coefficient (β) of the end‐diastolic pressure‐volume relationship (EDPVR) was calculated, which increased significantly in the TAC/DOCA group but was normalized after catestatin treatment (Figure 1I). Consistent with the echocardiographic parameter, LV end‐systolic pressure was elevated in the TAC/DOCA group but not in the TAC/DOCA+catestatin mice (Figure 1J), reflecting the exertion to maintain cardiac output under increased afterload. In addition, lower maximum LV pressure change ratio (dP/dt max; Figure 1K) and minimum LV pressure change ratio (−dP/dt min; Figure 1L) in TAC/DOCA mice demonstrated a prolonged ejection period and impaired LV relaxation, respectively. Thus, results from the pressure‐volume analysis further supported that TAC/DOCA mice exhibit slowed LV relaxation and increased LV chamber stiffness, while catestatin treatment effectively improved LV relaxation and compliance.

Catestatin Attenuates Cardiac Remodeling in Mice With HFpEF

Given the evident diastolic function change observed after catestatin treatment, we sought to determine catestatin effects on cardiac remodeling under TAC/DOCA challenge. M‐mode echocardiographic analysis demonstrated that the TAC/DOCA method increased the interventricular septum and LV posterior wall thickness dramatically at 7 weeks after surgery (Figure 2A through 2C). Concordant with the physiological results, increased end‐systolic diameter and reduced LV end‐diastolic diameter were recorded in TAC/DOCA mice, indicating less blood volume during diastolic filling (Figure 2D and 2E). Notably, the detrimental LV concentric hypertrophy was attenuated after catestatin therapy, with thinner wall thickness and more sufficient diastolic filling volume.

Figure 2. CST attenuates cardiac remodeling in mice with heart failure with preserved ejection fraction.

A, Representative M‐mode echocardiography images. B through E, Echocardiographic analysis of IVSd (B), LVPWd (C), LVIDs (D) and LVIDd (E) was measured. F, Representative immunofluorescent images of left ventricular wall tissue at diastasis (blue, DAPI; red, α‐actinin; green, laminin; scale bar, 50 μm). G through I, Comparison of cardiomyocytes dimension in single cell length (G), width (H), and surface area (I) (n=100 cell average dimension from 5 hearts per group). J and K, Percentage of cardiac fibrosis area (J) measured by sirius red staining (K) (n=5 per group from 5 random fields per heart; scale bar, 50 μm). Data are presented as mean ± SD and analyzed using 1‐way ANOVA followed by Tukey multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001 vs sham group; ^# P<0.05, ^## P<0.01, ^### P<0.001 vs TAC/DOCA group. CST indicates catestatin; DOCA, deoxycorticosterone acetate; IVSd, diastolic interventricular septum; LVIDd, end‐diastolic diameter; LVIDs, left ventricular end‐systolic diameter; LVPWd, diastolic left ventricular posterior wall thickness; and TAC, transverse aortic constriction.

After heart fixation at diastasis, the enlargement of LV cardiomyocytes was studied on a cellular level using α‐actinin to label sarcomeres and laminin to label extracellular matrix (Figure 2F). Consistent with the therapeutic effect observed at the LV chamber level, cardiomyocytes of TAC/DOCA+catestatin mice were smaller in both longitudinal and radial dimensions compared with TAC/DOCA mice (Figure 2G through 2I). These cell studies further supported an attenuated hypertrophic response after catestatin treatment. Furthermore, picrosirius red staining of the LV section was used to quantify the myocardial fibrosis, which is a major contributor to diastolic dysfunction. TAC/DOCA mice showed severe collagen deposition in percent LV area, whereas these changes were rescued by catestatin (Figure 2J and 2K). Together, these findings all supported that catestatin protects adverse cardiac remodeling in TAC/DOCA‐induced HFpEF mice.

Catestatin Downregulates Mitochondrial Oxidative Phosphorylation System Components in Mice With HFpEF

To further explore the underlying mechanism of catestatin anti‐HFpEF effect, transcriptomic analysis was performed in TAC/DOCA and TAC/DOCA+catestatin mice hearts at 7 weeks after surgery. A total of 1130 differentially expressed genes were identified, of which 709 transcripts were downregulated and 421 transcripts were upregulated after catestatin treatment (Table S2). Gene ontology analysis with downregulated transcripts in gene ontology pathways were enriched in mitochondrial complex I assembly, oxidation–reduction process, oxidative phosphorylation, and ETC (Figure 3A). Further analysis of top modified gene ontology pathways highlighted multiple genes involved in mitochondrial aerobic respiration being selectively targeted (Figure 3B). Moreover, the volcano plot demonstrated that expression of multiple genes coding the oxidative phosphorylation system components was significantly reduced by catestatin treatment (Figure 3C). Mitochondrial ETC is a major site of ROS production, an increase of which has been proved recently to contribute to diastolic dysfunction in HFpEF. ⁸ Complex I is central to mitochondrial oxidative stress by catalyzing electron transfer from NADH to ubiquinone coupled to proton transfer across the mitochondrial inner membrane, ¹⁶ the first step in the respiratory chain. Gene set enrichment analysis with all detected mRNAs also verified that NADH to cytochrome oxidase electron transfer chain and aerobic respiration pathway was significantly downregulated in TAC/DOCA+catestatin mice (Figure 3D). Collectively, these findings suggested mitochondrial ROS generation may be reduced by catestatin in mice with HFpEF.

Figure 3. CST downregulated mitochondrial oxidative phosphorylation system components in mice with heart failure with preserved ejection fraction.

A, GO analysis of RNA‐sequencing data from TAC/DOCA and TAC/DOCA+catestatin hearts. B, Chord diagram of top modified GO pathways demonstrating multiple genes involved in mitochondrial aerobic respiration was targeted. C, Volcano plot showing significantly decreased (green) and increased (red) genes with oxidative phosphorylation system components highlighted. D, Gene set enrichment analysis of “NADH to cytochrome oxidase electron transfer chain” and “aerobic respiration” pathways with all detected mRNAs. CST indicates catestatin; DOCA, deoxycorticosterone acetate; FDR, false discovery rate; GO, gene ontology; MIM, mitochondrial inner membrane; NES, normalized enrichment score; ROS, reactive oxygen species; and TAC, transverse aortic constriction.

Catestatin Restores Mitochondrial Respiratory Chain in Myocardium of Mice With HFpEF

To validate whether catestatin protected against HFpEF by restoring mitochondrial abnormality and attenuating oxidative stress, we first assessed the protein expression of ETC complex subunits in vivo. As expected, the expression of ETC complexes, especially complex I represented by subunit NDUFB8, was dramatically increased in TAC/DOCA hearts compared with sham groups (Figure 4A and 4B). Meanwhile, the expression of ETC complexes were markedly decreased after catestatin administration compared with the TAC/DOCA group. Because dysregulation of ETC components could cause abnormality in mitochondrial homeostasis, the mitochondrial morphology was evaluated using transmission electron microscopy. TAC/DOCA resulted in fewer disorganized mitochondria in abnormal shapes, whereas TAC/DOCA+catestatin displayed a normal mitochondria morphology similar to sham groups (Figure 4C). Detailed quantification analysis of the images demonstrated that mitochondria number was decreased in TAC/DOCA myocardium relative to other groups (Figure 4D). Slightly enlarged mitochondria size with sparse cristae was observed in TAC/DOCA hearts, which was reversed after catestatin treatment (Figure 4E and 4F). Further sphericity analysis showed catestatin effectively rescued TAC/DOCA‐induced heterogeneity in mitochondrial shapes (Figure 4G). Moreover, real‐time mitochondrial respiration capacity was evaluated in isolated primary cardiomyocytes from each group. Extracellular flux analysis demonstrated that basal, ATP production‐coupled, and the maximal respiration capacity were increased after TAC/DOCA surgery and restored after catestatin administration (Figure 4H). Following injection of oligomycin, a complex V inhibitor, we measured the ATP production‐independent basal oxygen consumption rate, which was known as proton leak and linked with ROS generation. ¹⁷ Importantly, treatment of catestatin reduced proton leak significantly in TAC/DOCA mice hearts, suggesting attenuation of mitochondrial oxidative stress (Figure 4I). As expected, mitochondrial complex I activity decreased dramatically after catestatin intervention compared with TAC/DOCA group (Figure 4J). Consistent with the critical contribution of mitochondrial ETC to ROS generation and restoration of ETC components by catestatin, catestatin induced a reduction of ROS in mice with HFpEF, demonstrated by lipid peroxidation fluorescent staining (Figure 4K and 4L). In aggregate, these findings suggested catestatin restored mitochondrial ETC components in mice with HFpEF and reduced mitochondrial oxidative stress.

Figure 4. CST restored mitochondrial respiratory chain in myocardium of mice with heart failure with preserved ejection fraction.

A, Representative immunoblotting images of mitochondrial respiratory electron transport chain complex subunits in heart tissue. B, Summary data of the relative complex I protein expression in all groups (n=6 per group). C, Representative transmission electron microscopy images of cardiac tissue from all groups (scale bar, 2 μm; 500 nm for zoomed). D through G, Quantification of average number of mitochondrial (D), mitochondrial area (E), mitochondrial cristae density per μm (F), and mitochondrial circularity (G) was analyzed (n=average results of 400 mitochondria from 5 hearts per group). H, Real‐time mitochondrial respiration capacity monitoring the oxygen consumption rate in primary cardiomyocytes isolated from each group (n=5 per group). I, The proton leak calculated as the remaining basal respiration not coupled to ATP production after oligomycin (n=5 per group). J, The complex I activity in myocardium of each group (n=5 per group). K and L, Representative image (K) and summary fluorescence intensity (L) of C11 BODIPY 581/591 staining of myocardium for lipid peroxidation and ROS formation (n=5 per group from 5 random fields per heart; Scale bar, 50 μm). Data are presented as mean ± SD and analyzed using 1‐way ANOVA followed by Tukey multiple comparisons test. *P<0.05, ***P<0.001 vs sham group; ^# P<0.05, ^## P<0.01, ^### P<0.001 vs TAC/DOCA group. CST indicates catestatin; DOCA, deoxycorticosterone acetate; FCCP, carbonyl cyanide‐4 (trifluoromethoxy) phenylhydrazone; Oligo, oligomycin; R/A, rotenone and antimycin A; and TAC, transverse aortic constriction.

Excessive Oxidative Stress Diminishes Catestatin Treatment Effects in HFpEF‐Like Cardiomyocytes

Because regulation of ETC complex I and decrease of mitochondrial oxidative stress by catestatin have been found in HFpEF, we examined whether catestatin protected against HFpEF through improvement of mitochondrial redox balance. Different concentrations of catestatin were added to the culture of a reported HFpEF‐like cell model ¹⁸ by treating the H9c2 cardiomyocytes with isoproterenol and macrophage‐conditioned medium (Figure 5A). Catestatin effectively improved HF, as evidenced by decreased natriuretic peptide B mRNA expression (Figure 5B). Importantly, the therapeutic effect of catestatin was totally abolished by Ru360, which inhibited mitochondrial calcium flow and induced oxidative stress by disturbing ETC components. ¹⁹ The detrimental effects of Ru360‐mediated ROS generation were confirmed by dihydroethidium staining (Figure 5C and 5D). Furthermore, mitochondrial ETC abnormality, as measured by proton leak, was increased in HFpEF‐like cardiomyocytes, rescued by catestatin, and neutralized after Ru360 intervention (Figure 5E). Consistently, the catestatin‐mediated downregulation of ETC complexes expression were diminished by Ru360 significantly, especially for complex I and complex III (Figure 5F through 5H). We also evaluated the complex I activity and found marked decrease under catestatin treatment but not after Ru360 was added (Figure 5I). Taken together, these results supported catestatin protects against HFpEF via attenuating mitochondrial ETC ROS generation.

Figure 5. Excessive oxidative stress diminished CST treatment effects in HFpEF‐like cardiomyocytes.

A, Experimental illustration of adding catestatin to HFpEF‐like cell model by treating the H9c2 cardiomyocytes with isoproterenol and MCM. B, The mRNA expression of heart failure marker Nppb in H9c2 cell with indicated treatments (n=6 per group). C and D, Representative image (C) and summary fluorescence intensity (D) of DHE staining of H9c2 cell with indicated treatments (n=6 per group from 5 random fields; Scale bar, 50 μm). E, The proton leak of H9c2 cell, calculated as the remaining basal respiration not couple to ATP production after oligomycin in real‐time mitochondrial respiration capacity monitoring (n=6 per group). F, Representative immunoblotting images of mitochondrial respiratory electron transport chain complex subunits in H9c2 cell with indicated treatments. G and H, Summary data of the relative complex III (G) and complex I (H) protein expression in all groups (n=4 per group). I, The complex I activity in H9c2 cell of each group (n=6 per group). Data are presented as mean ± SD and analyzed using 1‐way ANOVA followed by Tukey multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001 vs vehicle treated group; ^# P<0.05, ^## P<0.01, ^### P<0.001 vs MCM treated group; ^&& P<0.01, ^&&& P<0.001 vs MCM + 1μM catestatin‐treated group; ^@@ P<0.01, ^@@@ P<0.001 vs MCM + 10 μM catestatin‐treated group. CST indicates catestatin; DHE, dihydroethidium; HFpEF, heart failure with preserved ejection fraction; LPS, lipopolysaccharide; MCM, macrophage‐conditioned medium; and Nppb, natriuretic peptide B.

Serum Catestatin Level Is Elevated in Patients With HFpEF

Having found catestatin protects against diastolic dysfunction by reducing mitochondrial ROS generation in experimental models, we set out to explore its clinical relevance in patients with HFpEF. The baseline characteristics of all participants are included in Table S3. Serum catestatin level was significantly higher in patients with HFpEF relative to those non‐HF controls (11.21 [interquartile range, 6.81–19.12] ng/mL versus 23.62 [interquartile range, 11.53–34.81] ng/mL; P < 0.001; Figure 6A). Moreover, serum catestatin was positively correlated with NT‐proBNP (N‐terminal pro‐B‐type natriuretic peptide) level (r=0.41; P < 0.001; Figure 6B) and E/e′ ratio (r=0.25; P=0.002; Figure 6C) in all participants, suggesting that catestatin elevation could be a relatively insufficient but compensatory endogenous mechanism for HFpEF.

Figure 6. Serum CST level is elevated in patients with HFpEF.

A, Serum catestatin concentration in NHF controls (n=76) and HFpEF patients (n=81). B and C, Serum catestatin level was positively correlated with serum NT‐proBNP level (B) and E/e′ ratio (C) in all participants. Data are presented as mean ± SD and analyzed using Mann–Whitney U test in (A) or Spearman rank correlation analysis in (B and C). ***P<0.001 between indicates comparison. CST indicates catestatin; HFpEF, heart failure with preserved ejection fraction; NHF, non‐heart failure; and NT‐proBNP, N‐terminal pro‐B‐type natriuretic peptide.

Discussion

Here, we demonstrated that exogenous administration of recombinant catestatin protects against diastolic dysfunction, attenuates ventricular hypertrophy, and reduces myocardial fibrosis in TAC/DOCA‐induced mice with HFpEF. The therapeutic effects of catestatin on HFpEF are achieved by restoring mitochondrial ultrastructure abnormality, reducing mitochondrial ROS generation through suppression of proton leak from the ETC components dysregulation (Figure 7). Using a cohort of patients with HFpEF, we further found that the serum catestatin level is elevated in HFpEF and is related to the severity of diastolic dysfunction, indicating the clinical relevance of our finding.

Figure 7. A schematic illustration of CST alleviating HFpEF through reducing mitochondrial ROS generation from electron transport chain.

CST indicates catestatin; DOCA, deoxycorticosterone acetate; ETC, electron transport chain; HFpEF, heart failure with preserved ejection fraction; ROS, reactive oxygen species; and TAC, transverse aortic constriction.

HFpEF is now recognized as a complex, heterogeneous, multiorgan syndrome, involving cardiac, vasculature, immune, adipose, and other components. ²⁰ Conventional guideline‐directed therapies targeting the renin‐angiotensin‐aldosterone system failed to improve clinical outcome in HFpEF. ²¹ Catestatin has emerged as a pleiotropic neuroendocrine peptide, participating in the regulation of endovascular, inflammatory, and metabolic homeostasis. ²² The production of catestatin from prohormone chromogranin A can be carried out directly on cardiomyocyte membrane by extracellular proteases in the matrix. ²³ Ablation of macrophage‐produced catestatin in mice resulted in blood pressure elevation and cardiac macrophage infiltration with increased proinflammatory cytokines. ³ Exogenous catestatin was reported to alleviate cardiac hypertrophic responses by counterregulating β‐adrenergic stimulation. ²⁴ , ²⁵ In the aspect of metabolism, chromogranin A knockout mice exhibited increase in mitochondrial volume and overexpression of ETC complexes encoded genes, leading to hyperinsulinemia and an obesity phenotype under regular diet. ²⁶ Moreover, catestatin supplementation suppresses hepatic gluconeogenesis, improves insulin sensitivity, and enhances fatty acid oxidation in the liver. ²⁷ Our data indicated that catestatin treatment alleviates diastolic dysfunction, improves LV stiffness and compliance, and attenuates adverse cardiac remodeling in TAC/DOCA mice via reducing ROS generation from mitochondrial ETC electron leakage.

In the CATSTAT‐HF (Serum Catestatin Expression and Cardiometabolic Parameters in Patients With Congestive Heart Failure) study, researchers reported serum catestatin level positively correlates with soluble suppression of tumorigenicity 2 level in patients with acute worsening of HF, which might implicate the autonomic negative feedback inhibition of the sympathetic system during progressive cardiac remodeling. ²⁸ , ²⁹ Additionally, the plasma catestatin concentration was paralleled with the symptom burden of patients with chronic HF, as it increased among New York Heart Association classes I to IV. ³⁰ Reduced chromogranin A‐to‐catestatin conversion rate was found to be associated with increased mortality in acute HF, demonstrating a functional relevance of catestatin level in HF. ⁵ However, detailed pathophysiologic value of the elevated catestatin levels in HF remains unknown. To our knowledge, the present study first demonstrated the cardioprotective mechanism of catestatin in HFpEF under a preclinical setting. Furthermore, we confirmed an elevated serum catestatin level in our specific HFpEF cohort and demonstrated a mild correlation between catestatin and E/e′ ratio, suggesting a disease severity–dependent compensatory role of catestatin in HFpEF.

Cardiomyocyte mitochondrial structural and energetic abnormalities were observed during HFpEF development. ³¹ , ³² Mitochondrial damage could result in mismatch of ETC components and an increase in ROS generation, which subsequently activated downstream pathways, causing cardiac remodeling and diastolic dysfunction. ³³ Increase in cardiomyocyte ROS production was found in metabolic syndrome‐related HFpEF rats. ³⁴ Elevated myocardial lipid peroxidation and H₂O₂ levels also occurs in HFpEF patients. ³⁵ Recently, derivatives of reactive oxidative metabolites were reported to increase and positively correlated with HF‐related events in HFpEF, ³⁶ suggesting impact of oxidative stress on HFpEF outcomes. Moreover, administration of mitochondrial‐targeted antioxidant peptide SS‐31 markedly reduced angiotensin II–induced diastolic dysfunction, cardiac fibrosis, and apoptosis in mice. ³³ To validate whether catestatin improved diastolic function through reducing mitochondrial ROS generation from ETC proton leak, we employed Ru360 to disturb the oxidative phosphorylation complexes. As expected, Ru360 abrogated the protective effects of catestatin in HFpEF‐like cardiomyocytes, supporting the beneficial role of catestatin in HFpEF as a mitochondrial ETC modulator.

Several limitations remain in our study. First, only the classic TAC/DOCA mice were used as a HFpEF model, while various novel animal models mainly involving aging, nitrosative‐oxidative stress, and metabolic stress have been published. Second, catestatin could exert effects besides inhibiting mitochondrial oxidative stress in HFpEF, which was the focus of this work. For example, catestatin might promote the mitochondria dynamic by regulating cardiac macrophage homeostasis or directly modulate cardiomyocyte calcium transient. Third, a single‐center cross‐sectional HFpEF cohort was used, thereby allowing us to only preliminarily demonstrate a possible clinical relevance of catestatin rather than the prognostic value.

In conclusion, this study shed new light on how catestatin ameliorates HFpEF. We demonstrated catestatin supplementation improves diastolic function, cardiac fibrosis, and ventricular compliance in TAC/DOCA‐induced HFpEF mice by restoring mitochondrial ETC components and reducing ROS generation. Serum catestatin level was elevated in HFpEF patients and positively correlated with diastolic dysfunction reflected by E/e′ ratio, which indicates a feedback inhibition fashion of catestatin in HFpEF.

Sources of Funding

This research was supported by the National Natural Science Foundation of China (81970337 and 82270404), Program of Shanghai Academic/Technology Research Leader (21XD1402100), and Shanghai Sailing Program of Science and Technology Commission (23YF14360000).

Disclosures

None.

Supporting information

Data S1

Tables S1–S3

References ³⁷ , ³⁸ , ³⁹ , ⁴⁰ , ⁴¹