Elevated level of N-terminal pro B-type natriuretic peptide is associated with myocardial brosis in hypertrophic cardiomyopathy patients with preserved ejection fraction

Background: Myocardial brosis assessed by late gadolinium enhancement (LGE) on cardiovascular magnetic resonance (CMR) has been reported to be signicantly correlated with cardiovascular outcomes in hypertrophic cardiomyopathy (HCM) patients. However, data regarding non-invasive markers for detecting myocardial brosis were inconsistent and, not systematically evaluated in HCM patients with preserved ejection fraction (EF). Methods: In this study, 86 HCM patients with preserved EF and 33 controls were enrolled. The left ventricular function, end-diastolic maximum wall thickness (MWT), global systolic strains and extent of LGE (% LGE) were assessed. The biochemical indices were also recorded before the CMR examination. Results: Serum high-sensitivity cardiac troponin I (hs-cTnI) and N-terminal pro b-type natriuretic peptide (Nt-proBNP) levels were elevated in LGE-positive patients compared with LGE-negative patients (p < 0.05 for all). The LGE-positive patients had lower global longitudinal (GLS) and circumferential (GCS) strains than the LGE-negative group and the healthy controls (p < 0.05 for all). The LGE% was independently associated with the Nt-proBNP levels (standardized β = 0.627, p < 0.001), beta-blocker treatment (standardized β = -0.372, p = 0.01), MWT (standardized β = 0.481, p = 0.001) and GCS (standardized β = 0.406, p = 0.013). In the receiver operating characteristic (ROC) curve analysis, the combined parameters of Nt-proBNP ≥ 108 pg/mL and MWT ≥ 17.3 mm had good diagnostic performance for LGE, with a specicity of 81.3% and sensitivity of 70.0%. Conclusions: This study suggests that Nt-proBNP may be a biomarker associated with LGE% and, combined MWT, useful detecting brosis HCM and slice thickness: LGE of the LV long-axis and whole LV short-axis performed 10 to 15 after the of a bolus of gadolinium-diethylenetriamine pentaacetic acid using a phase-sensitive inversion The parameters as follows: FOV: 360 × 270 matrix: 256 ×192, TR/TE: 12.44 ms/1.19 inversion recovery time: 300 ip angle: 40°, and slice thickness:


Background
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy and its pathological features manifest as cardiac myocyte hypertrophy, disarray, and brosis [1,2]. Although, myocardial brosis is not so much a problem in itself, it is a feature of HCM with important clinical implications such as predisposition to sudden cardiac death (SCD) and progression to advanced disease [3,4]. Furthermore, myocardial brosis may be reversible and has been suggested as a powerful therapeutic target and prognosticator [5,6]. Extensive late gadolinium enhancement (LGE) imaging on cardiovascular magnetic resonance (CMR) is currently recognized as the gold standard for the identi cation of left ventricular (LV) focal replacement brosis [7]. Data currently suggest that LGE is highly promising in predicting SCD and progression to heart failure in HCM [8,9]. However, the administration of contrast agents needed for this technology may result in systemic nephrogenic sclerosis [10]. Therefore, identifying the non-invasive biomarkers for the early detection and prediction of myocardial brosis could have a role in management and risk strati cation in HCM.
Currently, cardiac-speci c biomarkers, particularly N-terminal pro b-type natriuretic peptide (Nt-proBNP) and high-sensitivity cardiac troponin I (hs-cTnI), have played a key role in the diagnosis, treatment and risk strati cation in the cardiovascular eld [11][12][13]. The two biomarkers are widely used in daily clinical practice due to their easy acquisition, cost-effectiveness, high reproducibility and no contraindications.
Although some modest sized studies have been showed a correlation of brosis with hs-cTnI and Nt-proBNP [14][15][16], the prognostic value of both biomarkers in predicting brosis in HCM patients was inconsistent and, not systematically evaluated in HCM patients with preserved ejection fraction (EF).
Additionally, CMR tissue-tracking technology can evaluate myocardial contractile abnormalities with rapid post-processing for routine cine images [17], which was widely used in various cardiovascular diseases [18][19][20]. There are some studies showing close correlation between myocardial mechanics and brosis in HCM, as well as associations with ventricular arrhythmias [21,22]. Thus, we speculate that the combination of clinical biomarkers and non-invasive CMR technology may be better utilized to predict myocardial brosis and, help clinicians to identify patients with poor prognosis at an early stage.
Our study, therefore, used advanced CMR tissue tracking technology and LGE analysis to explore the correlation between LGE and serum hs-cTnI levels, seurm Nt-proBNP levels, myocardial strains and wall thickness in HCM patients with preserved EF; we further investigated the diagnostic performance of these indexes for the detection of myocardial brosis as represented by LGE on CMR.

Study population
We

Laboratory measurements
The biochemical indices included the serum Nt-proBNP, hs-cTnI, creatine kinase (CK), creatine kinase-MB (CK-MB), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels, which were obtained for clinical evaluation purposes. Peripheral venous blood samples were collected from all HCM patients at the morning of CMR examination. Blood samples were centrifuged at 3000 rpm for 15 min, and plasma was stored at −80 ∘ C for further analysis. An electrochemiluminescent immunoassay assay (Roche Diagnostics, Mannheim, Germany) was performed for measurement of plasma Nt-proBNP levels. The analytical range was 5 to 35000 pg/mL and the normal reference range was ≤ 100 pg/mL. The interassay and intra-assay coe cients of variation were < 4.7% and < 5.8%, respectively. Serum levels of hs-cTnI were measured using the Abbott Architect high-sensitivity cTnI assay (Abbott Diagnostics, Abbott Park, USA). The lower limit of detection was 1.2 ng/L; the 99th percentile cutoff value was 26 ng/L; and the coe cient of variation was < 10%. Serum CK, CK-MB, AST and LDH levels were determined using an automatic particle chemiluminescence immunoassay (Abbott Aeroset, Minnesota) with the use of commercial kits (Abbott). The normal reference ranges of the assays were the following: 38 to 174 U/L for CK, < 6.6 ng/mL for CK-MB, 8 to 40 U/L for AST and 109 to 245 U/L for LDH.

CMR examination protocol
All CMR examinations were performed with a 1.5 T system (MAGNETOM Aera, Siemens Healthineers, Erlangen, Germany). The LV long-axis (4-, 3-, 2-chamber) and short -axis (covering all basal to apex segments) cine images were acquired using a balanced steady state free precession (bSSFP) sequence.

CMR image analysis
All CMR image analyses were semi-quantitatively performed using the commercial post-processing software (Cvi42, Circle Cardiovascular imaging, Calgary, AB, Canada).
For the quanti cation of LV volume and function, we imported all short-axis cines into the software and then manually delineated the LV endocardial and epicardial contours. All cardiac functional parameters were indexed to the body surface area (BSA) in this study. The LVEF, end-diastolic volume index (EDVI), end-systolic volume index (ESVI), stroke volume index (SVI), cardiac index and end-diastolic myocardial mass (MI) were acquired. Additionally, the LV end-diastolic maximum wall thickness (MWT) was de ned as the greatest segments of the 16-segment model of the American Heart Association (AHA). For the quanti cation of LV myocardial strain, we imported all LV long-and short-axis cines into the software.
Then, we manually delineated the LV endocardial and epicardial contours of all the above images in the end diastole stage. Then, the LV 3D global peak systolic longitudinal strain (GLS), circumferential strain (GCS) and radial strain (GRS) were semi-quantitatively calculated using the tissue feature tracking method ( Fig. 1).
For the quanti cation of the extent of LGE, we imported the whole LV short-axis slices of the LGE images into the software and manually delineated the LV endocardial and epicardial contours. A semiquantitative grey-scale threshold method was used to calculate the extent of LGE. The enhanced myocardium was de ned as a signal intensity threshold of > 6 SD above the mean signal intensity of the normal myocardium [23,24]. The previous studies demonstrated that the 6SD is the most optimal threshold especially for quantitative LGE in patients with HCM, as it is most strongly correlated with histopathology [23], as well as manual measurements [24]. Then, the total LV enhanced volume and mass were calculated, and the extent of LGE was expressed as the percentage of the total myocardial mass (%LGE) (Fig. 2). The HCM patients were divided into two subgroups based on the presence or absence of LGE. All papillary muscles and trabeculae were excluded from the LV myocardium during the LV function, deformation and LGE analyses.

Statistical analysis
The Kolmogorov-Smirnov test was used to check normality. Data are expressed as the mean ± SD or number (percentage) for all continuous and categorical variables. Differences between two groups were assessed using an independent-sample Student's t test or the Mann-Whitney test. Comparisons between three groups were analyzed using one-way ANOVA or the Kruskal-Wallis test, and the Bonferroni correction was selected as the post hoc test when appropriate. The chi-square test or Fisher's exact test was used for the comparison of all categorical variables. Pearson's or Spearman's correlation test was applied for the assessment of the LGE% and all candidate variables, as appropriate. Univariate and multivariate linear regression analyses were utilized to assess the associations between the LGE% and all candidate variables. Receiver operating characteristic (ROC) curve analysis was applied for the detection of the diagnostic performance of the presence of LGE. The highest Youden's index values were used to calculate the optimal cut-off values of the candidate variables. The sensitivity, speci city, optimal cut-off value, positive predictive value (PPV) and negative predictive value (NPV) were calculated and expressed with the corresponding 95% con dence intervals (CI). An intra-class correlation coe cient (ICC) with 95% CI was used for the assessment of the intra-and inter-observer agreement. A two-sided p value < 0.05 was considered statistically signi cant. Analyses were performed using SPSS Statistics (SPSS, version 21, IBM, Chicago, IL, USA) and MedCalc 16.2.0 (MedCalc Software, Mariakerke, Belgium).

Results
Baseline and biochemical characteristics subjects without LGE). The LGE-negative patients had a signi cantly higher heart rate than the controls and LGE positive patients (p < 0.01). The serum hs-cTnI and Nt-proBNP levels were signi cantly higher in LGE-positive patients than in LGE-negative patients (p < 0.01 for all) (Fig. 3, A and B). The LGE-positive patients had a signi cantly higher serum CK-MB levels than the LGE-negative group (p < 0.05 for all). There were no signi cant differences in any other characteristics of the study population, which are listed in Table 1. creatine kinase-MB, Nt-proBNP N-terminal pro b-type natriuretic peptide, hs-cTnI high-sensitivity cardiac troponin I. Table 2 shows the CMR parameters in HCM patients and the healthy controls as well as in the HCM subgroups strati ed by the presence of LGE. The LVEF of all HCM patients was more than 50% (range from 50.83% to 77.73%). Among the 48 LGE-positive patients, the mean LGE% was 10.22 ± 6.24%. Based on the LV myocardial systolic strain analysis, all HCM patients had a signi cantly lower GLS, GCS and GRS than the healthy controls (p < 0.05 for all). Additionally, the GLS and GCS were signi cantly lower in the LGE-positive patients than the LGE-negative group and the healthy controls (p < 0.05 for all). However, there were no signi cant differences in GLS, GCS and GRS between the LGE-negative patients and the healthy controls. The differences in any other LV volume and function parameters are shown in Table 2. The p and p' values reflect comparisons between 2 groups (controls. Vs. total) and 3 subgroups (controls. Vs.
LGE (- Correlations of LGE% with clinical and CMR parameters. The results of the univariate and multivariate regression analysis of the LGE% and the baseline clinical and CMR characteristics in HCM patients are described in Table 3. The LGE% in HCM patients was inversely associated with the use of beta-blockers and the GCS and was associated with an increased serum Nt-proBNP level and a greater MWT (p < 0.05 for all) (Fig. 3, C-F). Furthermore, any candidate variables with p < 0.3 and without collinearity on the univariate analysis were chosen for inclusion in the multinomial linear regression analysis using a stepwise algorithm model. The independent determinants of the LGE% were the serum Nt-proBNP level (standardized β = 0.672, p < 0.001) and MWT (standardized β = 0.481, p = 0.001). The use of beta-blockers (standardized β = -0.372, p = 0.010) and GCS (standardized β = 0.406, p = 0.013) were also independently correlated with the LGE% (Table 3).  and an NPV of 74.72% (Fig 4).

Discussion
The present results demonstrated that serum levels of Nt-proBNP and hs-cTnI were signi cantly higher in LGE-positive HCM patients with preserved EF, and the elevated levels of serum Nt-proBNP were independently associated with the LGE%. The combination of Nt-proBNP ≥ 108 pg/ml and MWT ≥ 17.3 mm had good diagnostic performance for the detection of LGE on CMR. Additionally, GLS and GCS were signi cantly decreased especially in LGE-positive group, and the impaired GCS was independently correlated with the LGE%. Moreover, the use of beta-blockers was correlated with a lower extent of myocardial brosis measured by LGE on CMR.
It is now well established that the serum Nt-proBNP and hs-cTnI levels were elevated in LGE-positive patients than in LGE-negative patients [14,16,25,26]. However, few data are available regarding the two biomarkers' utility for detecting LGE in HCM patients, and the prognostic value of both biomarkers in predicting brosis was inconsistent [14][15][16]. One study demonstrated that only the hs-cTnI was an independent indicator of the presence of LGE [14]. On the contrary, another study revealed that Nt-proBNP, not hs-cTnI, was strongly correlated with the amount of LGE [15]. And the present study not only showed that the serum levels of Nt-proBNP were correlated with the LGE% in multivariate analyses, but also was useful for the detection of myocardial brosis. The above differences in the relationship between the circulating biomarkers and the LGE% may be due to the different study populations and methods of quantifying LGE with CMR. Speci cally, in line with the study by Kawasaki et al. [16] our study excluded patients with LVEF < 50%, whereas the other two studies did not [14,15]. Additionally, previous studies usually used the visual scoring or semiquantitative scoring method [14,27], and the 2SD thresholding method was used in Kawasaki's study [16]. The current study used a semi-quantitatively grey-scale threshold method (6SD), previously shown to yield improved interobserver variability, reproducibility and precision with regards to LGE, as well as stronger correlations with histopathology in HCM patients [23,28].
Circulating Nt-proBNP is primarily produced by cardiomyocytes and is released in response to increased myocardial tension, stretching and neurohormonal activation [29]. However, the underlying mechanisms of the associations between elevated levels of Nt-proBNP and myocardial brosis in HCM patients are still under investigation. There are multiple possible explanations for the correlation between elevated Nt-proBNP and LGE. Previous research ndings implicated that myocardial brosis could promote diastolic dysfunction and abnormal microcirculation, leading to ischemia and replacement scarring [30][31][32].
Additionally, direct Nt-proBNP synthesis by cardiac broblasts, as an inhibitory anti brotic response via the extracellular signal-related kinase pathway has been demonstrated [33]. Thus, there is adequate pathophysiological background to consider investigating the potential of Nt-proBNP as a biomarker re ecting myocardial brosis.
Additionally, we also found that the LGE% was independently associated with MWT, which is consistent with the results of several previous studies [14,27,34] . As the presence of LGE could be observed especially in areas of ventricular hypertrophy in HCM patients [35], the correlation between MWT and myocardial brosis is considered reasonable. Moreover, a level of Nt-proBNP ≥ 108 pg/mL and MWT ≥ 17.3 mm had excellent diagnostic performance for the detection of LGE on CMR. These results suggest that the measurement of Nt-proBNP and MWT could be a non-invasive method of predicting myocardial brosis in HCM patients with preserved EF. Our ndings are expected to help clinicians to identify patients with poor prognosis at an early stage, especially for patients who cannot complete the LGE examination.
The present study also found that the GLS and GCS were signi cantly decreased, which was especially true in LGE-positive patients. Although the LVEF was normal or increased in the vast majority of HCM patients, the individual cardiac myocyte contractile and stretching forces were damaged and decreased, resulting in intrinsic dysfunction and myocardial remodeling [36,37]. Thus, myocardial systolic strains can detect cardiac dysfunction earlier than LVEF, especially in HCM patients with preserved EF. Additionally, we found that myocardial brosis was independently correlated with the GCS, while no correlation was observed with GLS or GRS. Some previous studies demonstrated that GLS was associated with the extent of LGE [38,39], while Erley et al. [40] found that LGE was correlated with GCS in HCM patients. A possible explanation of the above differences may be the differences in the postprocessing software used, the deformation acquisition techniques, study populations, clinical stages and medications in previous studies. The current study showed that the GCS is not only independently associated with LGE%, but also useful for detecting myocardial brosis based on ROC curve analysis, therefore suggesting its potential clinical utility for re ecting the presence of LGE.
In the present study, LGE negative patients had higher heart rate compared to both controls and LGE positive patients. Although beta-blocker use was similar in both HCM groups, dosage may have been different possibly explaining observed differences in heart rate. Additionally, the present results showed that the use of beta-blockers was signi cantly correlated with less myocardial brosis as measured by LGE on CMR. Beta-blockers therapy have proved effective in reducing myocardial ischemia and LVOT obstruction, and the current guidelines suggested these drugs as rst-line treatment in symptomatic patients with HCM, regardless of whether LV out ow obstruction exists [1,41]. The advantages of betablockers are mediated by sympathetic modulation of myocardial contractility, stiffness and heart rate, which can improve myocardial compliance and increase ventricular diastolic lling time [42,43]. However, to our knowledge, there are no studies demonstrating that beta-blockers treatment is associated with the amelioration of myocardial brosis in HCM patients. Whether beta-blockers therapy has a direct effect on the prevention, improvement or reversal of myocardial brosis needs to be further investigated in a longitudinal large-cohort study to determine whether there is a relationship between the duration of betablocker therapy, the order of beta-blocker therapy and HCM diagnosis, and the extent of LGE.

Limitations
There are several limitations in the present study. First, the sample size was relatively small. Second, this was a single-centre study, and the HCM subjects were selected with stringent criteria; therefore, some inherent biases were inevitable. Third, although LGE on CMR is limited to identifying diffuse myocardial brosis, this technique is widely and frequently used to assess myocardial brosis in different types of cardiovascular diseases. The 6SD thresholding method chosen in our study is the optimal method, especially for quantitative LGE in patients with HCM, as it is most strongly correlated with histopathology [23]. However, future large cohort studies with longer follow-up are needed to further con rm the parameters and predictors of the presence of LGE in HCM patients.

Conclusions
In conclusion, our results show that Nt-proBNP is a useful biomarker for detecting LGE and, combined with MWT, has good diagnostic performance for myocardial brosis in HCM patients with preserved EF.
Additionally, the decreased LV GCS was independently correlated with the LGE%, indicating its potential prognostic value for detecting myocardial brosis. These ndings suggesting that the combined noninvasive clinical biomarker and imaging technology can be a favorable alternative to LGE for assessing myocardial brosis in HCM patients when there were contraindications for contrast agent administration.

List Of Abbreviations
LGE Availability of data and materials The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Con ict of interest
The authors declare that they have no competing interests.

Funding
This study was funded by Hubei Province Key Laboratory of Molecular Imaging (02.03.2018-90) and Union Hospital, Huazhong University of Science and Technology (02.03.2019-101). The funders only provided funding and had no in uence on the study design, data collection, analysis, or interpretation, the decision to publish, or preparation of the manuscript.
Authors' contributions HSS, YC, YML and JL were responsible for the study concept, design and drafting the article. YML, JL, YKC XYZ and XYH were responsible for the data collection, statistical analysis and data interpretation. JG GZS and TTH were responsible for CMR image analysis. All the authors critically revised the manuscript and gave nal approval of the manuscript to be published.