Diagnostic potential of increased Klotho and FGF23 protein concentrations after myocardial infarction in patients with acute coronary syndrome

Abstract

Background

Klotho is a transmembrane and secretory protein and acts as a co-receptor for fibroblast growth factor 23 (FGF23). This study aimed to analyse the concentration of Klotho and FGF23 proteins in patients with myocardial infarction (MI).

Methods

The study group comprised 129 patients diagnosed with acute coronary syndrome (ACS), who were referred for further invasive diagnostics (MI group). Blood samples were collected at 4 time points: at admission, and 6 h, 24 h, and between 24–48 h post-admission. The criteria for the control subjects (n = 30) were no declaration of MI and ACS (non-MI group). Klotho and FGF23 concentrations in plasma were tested by ELISA at each time point.

Results

The concentration of soluble Klotho in the MI group was increased at admission, 6 h and 24 h post-admission, and then normalized at 24–48 h. Klotho concentration was also significantly increased in patients with ST-segment elevation MI (STEMI) only at admission, in comparison to non-ST-segment elevation MI (NSTEMI). The concentration of FGF23 in the MI group was higher at admission, 6 h and 24 h post-admission, and continued to increase after 24–48 h. There was an increase in FGF23 concentration in the STEMI group at 24–48 h post-admission, in comparison to NSTEMI.

Conclusions

The concentrations of Klotho and FGF23 in plasma were higher in patients with MI and changed over time. Thus, Klotho and FGF23 may be recognized as new factors in the diagnosis and/or monitoring of ACS, as well as novel therapeutic targets.

Introduction

Acute coronary syndrome (ACS) is a condition characterized by a sudden reduction in blood flow to the heart due to coronary occlusion, with signs and symptoms of sudden myocardial ischemia [1]. Signs and symptoms of ACS progress from unstable angina through non-ST segment elevation myocardial infarction (NSTEMI), where they are most intense in ST-segment elevation myocardial infarction (STEMI) [1]. Taking into account the complications of myocardial infarction (MI), early diagnosis and management of MI is essential [2].

Klotho is a transmembrane and secretory protein that was first identified in 1997 in transgenic mice. Primarily produced in the kidneys and brain, Klotho is also expressed in the heart and other tissues, and is a co-receptor for fibroblast growth factor 23 (FGF23) [3, 4]. It has been proven that Klotho participates in the reduction of oxidative stress, inflammation and fibrosis [5]. Recent studies have underlined the importance of Klotho in renal disorders, where Klotho deficiency served as an early biomarker and therapeutic target [6]. Klotho also acts as a renoprotective in acute kidney injury (AKI) [7]. Importantly, disrupted levels of Klotho and FGF23 are associated with cardiovascular disease (CVD) [8]. CVDs are responsible for a substantial portion of mortality, with estimates indicating that they account for about 30% of deaths in the EU and the USA [9]. Considering the morbidity and mortality of MI, there is an urgent need for the identification of novel markers and therapeutic targets in ACS. Thus, this study aimed to analyse the concentration of soluble alpha-Klotho (simply Klotho) and FGF23 proteins in patients with MI. The concentrations of Klotho and FGF23 proteins over time and with different types of MI (STEMI and NSTEMI) were evaluated.

Methods

Study subjects

This retrospective study consisted of 159 patients admitted by the Department and Clinic of Cardiology, Wroclaw Medical University between 2013 and 2015, and by the Department of Cardiology, University Hospital in Opole between 2019 and 2021. The study group (MI group) comprised 129 patients diagnosed with ACS, who were referred for further invasive diagnostics.

The control group (non-MI group), adjusted with age, was chosen from individuals invited by the Department and Clinic of Cardiology, Wroclaw Medical University (30 patients). Samples were sourced from the Biobank of Łukasiewicz Research Network — PORT Polish Centre for Technology Development (former Wroclaw Research Centre EIT+).

The protocol procedures were conducted following the Good Clinical Practice standards and the Declaration of Helsinki. The study was approved by the local Ethics Committee of Wroclaw Medical University: KB-54/2019 — samples from the Department and Clinic of Cardiology, Wroclaw Medical University; KB-514/2019 — samples from the Department of Cardiology, University Hospital in Opole; KB-387/2021 — samples from the Biobank of Łukasiewicz Research Network. Each ethics approval was obtained to ensure full compliance with institutional, national, and international ethical guidelines for biomedical research. Written informed consent was obtained from all the individuals for blood sample collection prior to participation.

Clinical data

Clinical data were obtained on admission and routine laboratory parameters were measured directly at the hospital’s central laboratory. The patient’s medical records and treatment protocol were completed by and obtained from the hospitals and used under the condition of maintaining the anonymity of the data.

Clinical material

Blood samples were collected by venepuncture of a cubital vein at 4 time points. Baseline sample was obtained at admission to the hospital, before invasive diagnostic (percutaneous coronary intervention; PCI). The other 3 samples were taken after the PCI procedure: 6 h, 24 h, and between 24–48 h post-admission. Each blood sample for Klotho and FGF23 concentration analysis was collected in tubes containing lithium heparin (68 IU) as an anticoagulant, were immediately centrifuged (2000 × g, 15 min, 20°C), and the plasma was separated, aliquot, and stored at −80° C.

Biochemical assays

Soluble α-Klotho concentration was measured with a solid-phase sandwich Alpha Klotho Human Soluble ELISA Kit #JP27998 (IBL Co. Ltd., Minneapolis, MN, USA), according to the manufacturer’s instructions. The concentration of FGF23 was determined using Human FGF23 ELISA Kit #orb390902 (Biorbyt Ltd., Cambridge, UK).

Statistical analysis

The data were analyzed with Statistica v. 13 (TIBCO Software Inc. (2017), Palo Alto, CA, USA), and the graphs were created with GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). To assess the normality of variance changes, the Shapiro–Wilk normality test was used for continuous variables in each group. For the comparisons of non-normally distributed data between two groups, the Mann–Whitney U test was used. For the comparison of means for normally distributed variables between two groups, the Student’s t-test was used. Pearson’s chi-squared test was used for the analysis of categorical data. The comparison of data between more than two groups was made with the nonparametric Kruskal–Wallis test with the post hoc test (Dunn’s multiple comparisons test). The correlation analysis was assessed with the Pearson’s or Spearman’s tests. The values of p < 0.05 were regarded as statistically significant.

Results

Basic clinical and biochemical data of patients in the study groups

129 patients were enrolled in total, 98 men and 31 women participated in the MI group respectively. Among them, 75 patients presented STEMI and 54 patients were NSTEMI. Non-MI group included a total of 30 patients, 16 men and 14 women. The demographic, clinical presentation, and laboratory data in MI and non-MI groups are shown in Table 1. Table 2 shows variables in STEMI and NSTEMI groups.

Table 1.

Basic clinical and biochemical data of patients with and without MI

	MI	Non-MI	P-value
Total number of patients	129	30
Age [years]	68 (60; 75)	71 (63; 84)	ns
Male, n [%]	98 (76.0)	16 (53.3)	0.013
LVEF [%]	47 (40; 55)	65 (60; 65)	< 0.001
Arrhythmias, n [%]	38 (29.5)	15 (50.0)	0.032
CAD, n [%]	62 (48.1)	3 (10.0)	< 0.001
CHF, n [%]	37 (28.9)	7 (23.3)	ns
CHD, n [%]	1 (1.2)	2 (6.7)	ns
AHD, n [%]	63 (75.0)	15 (50.0)	0.012
Myocarditis, n [%]	1 (1.2)	5 (16.7)	0.001
Obesity (BMI ≥ 30), n [%]	30 (23.3)	9 (30.0)	ns
DM, n [%]	46 (35.7)	7 (23.3)	ns
Hypertension, n [%]	86 (66.7)	19 (63.3)	ns
Atherosclerosis, n [%]	12 (9.5)	4 (13.3)	ns
Smoking, n [%]	55 (43.0)	3 (10.0)	< 0.001
DVT, n [%]	3 (3.6)	3 (10.0)	ns
PE, n [%]	3 (2.4)	4 (13.3)	0.009
eGFRmin [mL/min/1,73 m2]	70.6 ± 17.0	62.1 ± 16.4	0.019
Troponin Imax [pg/mL]	376.2 (11.8; 5345.3)	0.01 (0.0; 1.9)	<0.001
TC [mg/dL]	183.0 (150.0; 218.0)	182.5 (130.0; 234.0)	ns
LDL cholesterol [mg/dL]	119.0 (84.0; 146.0)	100.5 (77.0; 127.0)	ns
HDL cholesterol [mg/dL]	42.5 (36.0; 51.0)	47.0 (40.0; 57.0)	ns
TG [mg/dL]	122.0 (101.0; 169.0)	107.5 (82.0; 163.0)	ns
Glucosemax [mg/dL]	150.0 (121.0; 196.5)	117.5 (103.0; 135.0)	< 0.001
HbA1C [%]	5.9 (5.5; 6.7)	5.6 (5.3; 6.13)	0.032
hsCRPmax [mg/L]	17.7 (6.0; 62.7)	4.4 (1.8; 16.9)	0.006
Hb [g/dl]	14.0 (12.8; 15.3)	13.7 (11.9; 15.2)	ns
Ht [%]	41.5 (38.2; 44.0)	40.8 (36.4; 44.2)	ns
RBC [×106/μL]	4.5 ± 0.6	4.4 ± 0.6	ns
WBC [×103/μL]	10.6 ± 3.6	8.1 ± 2.6	< 0.001
PLT [×103/μL]	232.0 (192.0; 288.0)	205.0 (162.0; 268.0)	ns

Inclusion criteria for the MI group: age ≥ 18 years and confirmed diagnosis of STEMI or NSTEMI based on the clinical signs, electrocardiography and blood troponin level; exclusion criteria for the MI group: the presence of malignancy, severely decreased eGFR (< 30 mL/min/1.73 m²), kidney failure, AKI or CKD stage G4–5, to obviate potential interferences; the criteria for the control subjects were the same for the study group and no declaration of present or past MI, ACS, PCI or CABG, based on the examination conducted on admission; data expressed as numbers and percentages for categorical variables were analysed with Pearson’s chi-squared test, and for numerical variables as mean ± SD (for normally distributed variables) with Student’s t-test or the median + 25th and 75th percentiles (for non-normally distributed variables) with Mann–Whitney U test; ACS — acute coronary syndrome; AHD — acquired heart disease; AKI — acute kidney injury; BMI — body mass index; CABG — coronary artery bypass grafting; CAD — coronary artery disease; CHD — congenital heart defects; CHF — congestive heart failure; CKD — chronic kidney disease; DM — diabetes mellitus; DVT — deep vein thrombosis; eGFR — estimated glomerular filtration rate; Hb — hemoglobin; HbA1c — hemoglobin A1c; HDL — high-density lipoprotein; hs-CRP — high-sensitivity C-reactive protein; Ht — hematocrit; LDL — low-density lipoprotein; LVEF — left ventricular ejection fraction; MI — myocardial infarction; ns — not significant;NSTEMI — non-ST segment elevation myocardial infarction; PCI — percutaneous coronary intervention; PE — pulmonary embolism; PLT — platelet count; RBC — red blood cell count; STEMI — ST segment elevation myocardial infarction; TC — total cholesterol; TG — triglycerides; WBC — white blood cell count

Table 2.

Baseline characteristics of patients in STEMI and NSTEMI groups

	STEMI	NSTEMI	P-value
Total number of patients	75	54
Age [years]	68 (58; 75)	69 (63; 78)	ns
Male, n [%]	59 (78.6)	39 (72.2)	ns
LVEF [%]	45.6 ± 10.5	49.6 ± 12.8	ns
Arrhythmias, n [%]	19 (25.3)	19 (35.2)	ns
In-hospital PCI, n [%]	73 (97.3)	35 (64.8)	< 0.001
CAD, n [%]	35 (46.7)	27 (50.0)	ns
CHF, n [%]	20 (26.7)	17 (32.1)	ns
CHD, n [%]	0 (0.0)	1 (2.4)	ns
AHD, n [%]	33 (78.6)	30 (71.4)	ns
Myocarditis, n [%]	0 (0.0)	1 (2.4)	ns
Previous MI, n [%]	9 (13.9)	16 (29.6)	0.035
Previous PCI, n [%]	17 (22.7)	8 (14.8)	ns
Obesity (BMI ≥ 30), n [%]	18 (24.0)	12 (22.2)	ns
DM, n [%]	29 (38.7)	17 (31.5)	ns
Hypertension, n [%]	48 (64.0)	38 (70.4)	ns
Atherosclerosis, n [%]	7 (9.5)	5 (9.4)	ns
Smoking, n [%]	37 (49.3)	18 (34.0)	ns
DVT, n [%]	2 (4.8)	1 (2.4)	ns
PE, n [%]	3 (4.0)	0 (0.0)	ns
eGFRmin [mL/min/1,73 m2]	72.0 ± 17.2	69.1 ± 16.8	ns
Troponin Imax [pg/mL)	2013.0 (68.8; 10000.0)	54.8 (0.9; 538.1)	< 0.001
TC [mg/dL]	183.0 (150.0; 213.0)	182.5 (150.5; 228.5)	ns
LDL cholesterol [mg/dL]	119.0 (89.0; 139.0)	119.0 (81.5; 160.5)	ns
HDL cholesterol [mg/dL]	40.5 (36.0; 50.6)	44.3 (34.7; 52.0)	ns
TG [mg/dL]	117.5 (104.0; 160.0)	127.0 (100.0; 182.0)	ns
Glucosemax [mg/dL]	160.0 (130.0; 207.0)	129.0 (110.0; 190.0)	0.002
HbA1C [%]	6.0 (5.6; 6.8)	5.9 (5.4; 6.2)	ns
hsCRPmax [mg/L]	14.8 (5.2; 79.5)	19.2 (7.8; 59.6)	ns
Hb [g/dl]	14.2 (13.5; 15.5)	13.5 (12.5; 14.6)	0.014
Ht [%]	42.0 (39.5; 44.0)	40.1 (37.1; 43.3)	0.037
RBC [×106/μL]	4.5 ± 0.6	4.4 ± 0.7	ns
WBC [×103/μL]	11.7 ± 3.8	9.5 ± 3.0	0.005
PLT [×103/μL]	237.0 (191.0; 308.0)	229.5 (193.0; 259.0)	ns

Data expressed as numbers and percentages for categorical variables were analyzed with Pearson’s chi-squared test, and for numerical variables as mean ± SD (for normally distributed variables) with Student’s t-test or the median + 25th and 75th percentiles (for non-normally distributed variables) with Mann–Whitney U test; AHD — acquired heart disease; BMI — body mass index; CAD — coronary artery disease; CHD — congenital heart defects; CHF — congestive heart failure; DM — diabetes mellitus; DVT — deep vein thrombosis; eGFR, estimated glomerular filtration rate; Hb — hemoglobin; HbA1c — hemoglobin A1c; HDL — high-density lipoprotein; hs-CRP — high-sensitivity C-reactive protein; Ht — hematocrit; LDL — low-density lipoprotein; LVEF — left ventricular ejection fraction; MI — myocardial infarction; NSTEMI — non-ST segment elevation myocardial infarction; PCI — percutaneous coronary intervention; PE — pulmonary embolism; PLT — platelet count; RBC — red blood cell count; STEMI — ST segment elevation myocardial infarction; TC — total cholesterol; TG — triglycerides; WBC — white blood cell count

As compared with the non-MI group, there were more men among MI patients, and they showed significantly lower left ventricular ejection fraction (LVEF) and lower arrhythmia prevalence (Table 1). In comorbidities manifestation, the patients with MI showed a higher incidence of coronary artery disease (CAD), acquired heart disease (AHD), myocarditis and smoking, and lower incidence of pulmonary embolism. In terms of laboratory tests, estimated glomerular filtration rate (eGFR), troponin I (TnI), glucose, haemoglobin A1c (HbA1c), high-sensitivity C-reactive protein (hs-CRP) and white blood cell count (WBC) levels were higher in MI group, as compared to non-MI group (Table 1).

There was no significant difference between STEMI and NSTEMI groups in terms of demographic and clinical presentation, except for in-hospital PCI treatment and previous MI prevalence (Table 2). In-hospital PCI was performed in most STEMI patients; however, the rate of previous MI was higher in the NSTEMI group. STEMI patients showed significantly higher levels of TnI, glucose, haemoglobin (Hb), haematocrit (Ht) and WBC, compared with the NSTEMI group (Table 2).

The concentration of Klotho and FGF23

The concentration of soluble Klotho protein in the MI group was increased at admission, 6 h and 24 h after admission, and then normalized at 24–48 h after admission, as compared to non-MI patients (Fig. 1).

Figure 1.

The concentration of soluble Klotho protein in MI group at admission to the hospital (n = 72), and 6 h (n = 45), 24 h (n = 45) and 24–48 h (n = 57) after admission, and in non-MI group (n = 26); MI — myocardial infarction; boxes — 25–75% percentile, whiskers — min to max + median, Kruskal–Wallis test + Dunn’s multiple comparison test (post-hoc)

The concentration of FGF23 protein in the MI group was higher at admission, 6 h and 24 h after admission, and continued to increase at 24–48 h after admission, as compared to non-MI patients (Fig. 2).

Figure 2.

The concentration of FGF23 protein in MI group at admission to the hospital (n = 63), and after 6 h (n = 39), 24 h (n = 38) and 24–48 h (n = 55), and in non-MI group (n = 24); FGF23 — fibroblast growth factor 23; MI — myocardial infarction; boxes — 25–75% percentile, whiskers — min to max + median, Kruskal–Wallis test + + Dunn’s multiple comparison test (post-hoc)

There was an inverse correlation between Klotho concentration at 24–48 h after admission and hs-CRP (r = −0.34; p < 0.05), red blood cell count (RBC) (r = 0.33; p < 0.05), Ht (r = 0.35; p < 0.05), and Hb (r = 0.32; p < 0.05) (Fig. 3). There was no statistically significant correlation of FGF23 concentration at 24–48 h after admission with analysed parameters (Fig. 3).

Figure 3.

Correlation matrix among the selected biochemical parameters, and the concentrations of Klotho and FGF23 at 24–48 h after admission (t3). Correlations were obtained by deriving the Spearman correlation coefficient. Blue is a positive correlation, and red is a negative correlation. Color intensity is proportional to the correlation coefficients. *p < 0.05; FGF23 — fibroblast growth factor 23; Hb — haemoglobin; HbA1c — haemoglobin A1c; hs-CRP — high-sensitivity C-reactive protein; Ht — haematocrit; RBC — red blood cell count; TnI — troponin I; t3 — concentration at 24–48 h after admission

Klotho and FGF23 concentrations based on MI types

Klotho concentration was significantly increased in patients with STEMI only at admission, in comparison to NSTEMI patients (Fig. 4A). There was no significant difference in Klotho concentration in the STEMI group at 6 h (Fig. 4B), 24 h (Fig. 4C) and 24–48 h post-admission (Fig. 4D), compared to the NSTEMI group.

Figure 4.

The concentration of soluble Klotho protein in STEMI and NSTEMI groups at admission to the hospital (A), and after 6 h (B), 24 h (C) and 24–48 h (D); MI — myocardial infarction; boxes — 25–75% percentile, whiskers — min to max + median, Mann-Whitney test

The concentration of FGF23 did not differ significantly between the STEMI group at admission (Fig. 5A), 6 h (Fig. 5B) and 24 h (Fig. 5C) after admission, compared to the NSTEMI group. There was a statistically significant increase in FGF23 concentration in the STEMI group at 24–48 h post-admission (Fig 5D), in comparison to the NSTEMI group.

Figure 5.

The concentration of FGF23 protein in STEMI and NSTEMI groups at admission to the hospital (A), and after 6 h (B), 24 h (C) and 24–48 h (D); FGF23 — fibroblast growth factor 23; MI — myocardial infarction; boxes — 25–75% percentile, whiskers — min to max + median, Mann–Whitney test

An impact of comorbidities and treatment on Klotho and FGF23 concentrations in patients with MI

MI patients with CAD concomitance showed significantly lower concentration of Klotho at admission than individuals with no CAD (Fig. 6A). The reduction in Klotho concentration at admission was also observed in the MI with the congestive heart failure (CHF) group, compared to the MI and non-CHF group (Fig. 6B). The concentration of Klotho at 24–48 h post-admission in MI patients was also lower after treatment with an antiplatelet drug (Fig. 6C).

Figure 6.

(A) The concentration of Klotho at admission to the hospital in MI patients with or with no CAD; (B) The concentration of Klotho at admission in MI patients with or with no CHF; (C) An influence of treatment with antiplatelet drug (clopidogrel) on Klotho concentration after 24–48h in MI patients; (D) The concentration of FGF23 at admission to the hospital in MI patients with or without in-hospital PCI; (E) The concentration of FGF23 at admission in MI patients with or without prior PCI; (F) The influence of prior PCI on FGF23 concentration after 24–48 h (t3) in MI patients; CAD — coronary artery disease; CHF — congestive heart failure; FGF23 — fibroblast growth factor 23; MI — myocardial infarction; PCI — percutaneous coronary intervention; boxes — 25–75% percentile, whiskers — min to max + median, Mann–Whitney test

MI patients who were referred to in-hospital PCI showed a lower concentration of FGF23 at admission than the non-PCI group (Fig. 6D). Contrary, MI patients who have had PCI in the past showed a higher concentration of FGF23 at admission (Fig 6E) and at 24–48 h post-admission (Fig. 6F), than non-past PCI group.

Discussion

This study reveals increased concentrations of Klotho and FGF23 following MI, with temporal changes. Elevated Klotho concentrations were observed at admission, persisting for 6–24 hours before normalizing after 24–48 hours. Notably, STEMI patients showed a significant rise in Klotho only at admission, suggesting its potential as an early STEMI marker. FGF23 concentration remained elevated at all time points, with higher concentration in STEMI patients 24–48 hours post-admission. This research highlights the diagnostic potential of Klotho and FGF23 as biomarkers in MI, offering new insights into their dynamics and expanding understanding of CVD.

Research from the last decade identified Klotho as an early biomarker and therapeutic target in kidney injury [10, 11]. Since Klotho levels are known to be lower and FGF23 levels are higher in patients with renal dysfunctions, in the current study individuals with severely reduced eGFR, AKI, and chronic kidney disease (CKD) stage G4–5 were excluded to minimize the effect of these factors [12, 13]. Here we reported a higher concentration of soluble Klotho protein in patients with MI. The increase was observed at hospital admission and persisted 6 to 24 h post-admission. Then, the concentration of Klotho started to normalise after 24–48 h. Importantly, the concentration of Klotho was notably higher in the STEMI group compared to the NSTEMI group at admission. The temporal dynamics of Klotho differed between these two types of MI, with a more pronounced acute elevation in STEMI. This distinction is crucial as it suggests Klotho may serve as an early marker for STEMI and could help in the stratification and identification of patients at higher risk of severe cardiac events in the acute phase of MI. The elevated concentration of Klotho in the early phase of MI may represent a physiological response against myocardial failure during ACS based on increased de novo synthesis of Klotho and may indicate a compensative mechanism to promote repair processes in the heart. Alternatively, it also can result from the release of Klotho from damaged or stressed cells due to cellular damage mechanisms [14–15]. Distinguishing between de novo synthesis in cardiac or renal tissues and release from pre-existing stores in damaged cells requires further targeted investigation. Moreover, the higher Klotho concentrations in STEMI may reflect a more intense biological reaction to the larger area of myocardial injury, typically seen in STEMI compared to NSTEMI [1]. Its elevation in STEMI could represent an attempt to mitigate the more severe insult. Currently, only a few studies have focused on the expression of Klotho in humans immediately after MI, and no studies have compared Klotho concentrations between STEMI and NSTEMI patients over time. Pei et al. [16] reported an increased concentration of Klotho in patients with acute MI who developed AKI, compared to the non-AKI group. They proposed Klotho as a biomarker for AKI following acute MI. Serum Klotho level was also higher in older adults with a clinical history of MI. They suggested that serum Klotho may be an independent indicator of post-MI in elderly patients, potentially playing a role in secondary prevention due to its elevated levels in affected outpatients [17]. In another study, patients with CAD and lower Klotho levels were less likely to have prior MI [18]. Taneike et al. [12] reported elevated levels of Klotho in patients with HF, and in those who showed improvement after intensive treatment. After improvement, the Klotho level dropped to basal levels and it was proposed as a novel biomarker for treatment responsiveness. Contrary to the present observation, the concentration of plasma Klotho was reduced in patients with STEMI, as compared to healthy controls. However, the blood samples were taken within the first 24 hours of hospital admission, thus no dynamics of Klotho level changes over time were shown [19]. In this study, a negative correlation of Klotho with the level of CRP, and a positive correlation with RBC, Ht and Hb were also shown. Peng et al. [20] found a negative association between serum Klotho and CRP, suggesting a potential cardioprotective role of Klotho. Likewise, serum Klotho levels were inversely correlated with CRP in patients with established CVD [21]. Regarding the red blood cell parameters, Klotho deficiency was found to be characteristic during CKD with concomitance of anaemia and cardiovascular complications [22]. A positive correlation of serum Klotho level with Hb and RBC in middle-aged and older adults was also reported [23]. It was revealed that Klotho directly regulates the differentiation of hematopoietic stem cells, as well as the generation and maturation of erythroid cells in mice. They proposed negative feedback between Klotho and erythropoietin synthesis [24]. Taken together, all the above may suggest that increased concentrations of Klotho protein in patients after MI indicate a beneficial impact of Klotho to protect cardiac cells against ischemic injury and/or a compensatory mechanism to prevent cardiovascular complications.

Scientists reported a higher prevalence of CHF and MI in adults with a low concentration of serum Klotho, which could be reduced after the restoration of an adequate protein level [25]. Akhiyat et al. [26] showed lower levels of Klotho in patients with coronary microvascular disease in the absence of CAD or ACS and proposed Klotho as a biomarker and a therapeutic target. In the current research, Klotho concentration in MI patients with CAD and CHF concomitance was lower, as well as after treatment with an antiplatelet drug. Similarly, Cai et al. [27] and Jeinsen et al. [28] showed a lower Klotho concentration in CHF patients. Decreased concentrations of serum Klotho was observed also in individuals with CAD [29]. A reduced level of Klotho in patients with CAD was related to death or CHF hospitalization as well [18]. Importantly, scientists reported that higher levels of Klotho are related to reduced risk of death. In a cohort of elderly non-CKD individuals, the rate of all causes death was higher in the group with low Klotho levels [30]. Additionally, platelet count and lifespan were found to correlate with circulating Klotho, which could explain the influence of the antiplatelet drug on Klotho concentration in the present study [31]. However, these interactions need to be further investigated. Considering the results obtained in this study and previous reports, Klotho may protect against CVD, and restoration of its adequately high levels could be used as a beneficial factor in heart diseases.

It is known that FGF23 needs membrane-bound Klotho protein to react with the FGF receptor [5]. In this study, the concentration of FGF23 was increased at each time point in patients who have had MI. FGF23 was also higher in the STEMI group between 24 and 48 h post-admission, which was not observed for Klotho. It may suggest that FGF23 could be a late marker of this type of MI. The research on experimental MI in rat and mouse models showed upregulated levels of circulating FGF23 [32]. Similarly, Singh et al. [33] reported increased levels of FGF23 in patients after MI. Early elevation of FGF23 levels after MI was related to 1-year major cardiovascular events. Schmitz et al. [34] proposed FGF23 as a useful marker in the long-term treatment of STEMI patients and a possible target for drug development. In our study, FGF23 concentration continued to increase since the Klotho concentration normalised at 24–48 h after admission. Takahashi et al. [13] reported elevated levels of serum FGF23 in MI patients on the 5^th and 7^th day after PCI. It was negatively correlated with changes in left ventricular ejection fraction 6 months after onset. There was also an association of circulating FGF23 with the left ventricular remodelling or cardiogenic shock in MI patients [35, 36]. Importantly, elevated levels of FGF23 were a strong independent predictor of HF and death after ACS. Despite the lack of statistical significance, Klotho levels tended to be associated with a reduced incidence of studied outcomes [37]. Thus, cardiac and/or circulating FGF23, but not Klotho, might be an indicator of weak recovery of cardiac function and be involved in adverse changes in the heart tissue after MI.

The current report corroborates results from previous observational studies which proclaimed no or a weak correlation between circulating Klotho and FGF23 concentrations [18, 38]. In this research, patients further referred to in-hospital PCI showed lower concentrations of FGF23 at admission than the non-PCI group. Contrary, PCI in history and current MI influenced enhanced production of FGF23. Similarly, the highest concentration of FGF23 in STEMI patients who underwent PCI was observed after 1 year of follow-up [39]. FGF23 level was also increased in coronary heart disease (CHD) patients who underwent PCI with a risk of 2-year in-stent restenosis [40]. This may suggest a negative prognostic role of FGF23. Cornelissen et al. [41] reported the association of serum FGF23 level with one-year mortality in patients with MI and concomitant HF. In another study, high levels of systemic FGF23 were related to all-cause death and cardiovascular mortality in patients, independently of Klotho levels [42]. Numerous studies confirmed the upregulation of FGF23 in patients at risk of adverse cardiac events, including MI, LVH, AHF, arrhythmias, vascular calcification and stroke [43]. Unlike the Klotho level, FGF23 was higher in both, plasma and bone marrow, and was positively related to disease severity, progression and death during CHF [28, 38]. Similarly, patients with low levels of Klotho and high levels of FGF23 were at high risk for cardiovascular death or hospitalization for HF [18]. Taken together, an elevated level of FGF23 seems to be an unfavourable prognostic factor in CVDs and may be utilized in patient monitoring.

The study has some limitations including a small and unequal sample size in each group. The variable n number in individual study groups at different time points on the graphs results from an inability to determine Klotho and/or FGF23 concentrations in all tested samples (technical problems and/or no sample for analysis). Acknowledged herein was significant disparity in gender distribution between the control group and the post-MI group. This imbalance in gender composition has the potential in some measure to influence in the study outcomes and may affect the generalizability of the present findings. Previous research has demonstrated that gender differences can impact cardiovascular outcomes, and therefore, it is recognized that the unequal gender distribution in the current sample may somehow constrain the ability to draw definitive conclusions regarding the relationships observed in this study. In addition, protein concentrations during follow-up were not measured, and there is no data on their changes over a longer time. Therefore, in clinical practice, the results should be interpreted with caution.

The most important perspectives for future studies include elucidating the mechanisms of changes in Klotho and FGF23 concentrations during MI. Additionally, longitudinal research should explore the prognostic value of these proteins in predicting myocardial recovery and adverse outcomes. Finally, interventional studies targeting Klotho and FGF23 pathways could provide insights into their therapeutic potential in mitigating myocardial injury and promoting cardiac repair.

Conclusions

This study identifies increased concentrations of Klotho and FGF23 in MI patients, highlighting their potential as diagnostic markers and therapeutic targets for ACS.