A Development of Nucleic Chromatin Measurements as a New Prognostic Marker for Severe Chronic Heart Failure

Abstract

Background: Accurate prediction of both mortality and morbidity is of significant importance, but it is challenging in patients with severe heart failure. It is especially difficult to detect the optimal time for implanting mechanical circulatory support devices in such patients. We aimed to analyze the morphometric ultrastructure of nuclear chromatin in cardiomyocytes by developing an original clinical histopathological method. Using this method, we developed a biomarker to predict poor outcome in patients with dilated cardiomyopathy (DCM).

Methods and results: As a part of their diagnostic evaluation, 171 patients underwent endomyocardial biopsy (EMB). Of these, 63 patients diagnosed with DCM were included in this study. We used electron microscopic imaging of cardiomyocyte nuclei and an automated image analysis software program to assess whether it was possible to detect discontinuity of the nuclear periphery. Twelve months after EMB, all patients with a discontinuous nuclear periphery (Group A, n = 11) died from heart failure or underwent left ventricular assist device (VAD) implantation. In contrast, in patients with a continuous nuclear periphery (Group N, n = 52) only 7 patients (13%) underwent VAD implantation and there were no deaths (p<0.01). We then evaluated chromatin particle density (Nuc-CS) and chromatin thickness in the nuclear periphery (Per-CS) in Group N patients; these new parameters were able to identify patients with poor prognosis.

Conclusions: We developed novel morphometric methods based on cardiomyocyte nuclear chromatin that may provide pivotal information for early prediction of poor prognosis in patients with DCM.

Fig 1. Classification of Groups A and N Based on Quantitative Analysis.

(A) Representative original electron microscopic images before digital analysis are shown (10,000× magnification). The perinuclear condensed chromatin was isolated automatically using image analysis software. The area of perinuclear condensed chromatin, which is defined as the area with a grayscale value between 146 and 256, is outlined in red. Black arrows indicate areas of discontinuous nuclear periphery (discontinuous signals of perinuclear condensed chromatin by automatic detection). (B) Two types of nuclei, Group A and Group N, are shown. Representative conventional electron microscopic images (80 kV). Group A: These nuclei have discontinuous perinuclear condensed chromatin with irregular margins and unclear borders. There are homogeneous aggregations of particles in the nucleoplasm. Group N: These nuclei have continuous margins and clear borders. The granular particles in the nucleoplasm occasionally accumulate in spots, but do not aggregate closely. Scale bar: 1 μm. (C) High-power conventional electron microscopic (100 kV) images of chromatin structure in Patients 1 and 2, respectively. In Patient 1, the nucleus has an aggregated structure composed of poorly defined particles. In Patient 2, the nucleus has relatively sparse accumulations of particles less than 30 nm in size. Scale bar: 200 nm.

Fig 2. Representative Nuclei of 6 Patients from Each Group.

Representative images are shown for each group. Six patients from Group A (A)–(F). Six patients from Group N: (G)–(L). Scale bar: 2 μm.

Fig 3. Protocol to Quantitatively Measure Chromatin Area in Group N.

Our novel method is composed of the following steps. (A) Representative image prior to the availability of digital analysis (10,000× magnification). (B) The perinuclear condensed chromatin was isolated automatically by image analysis software. The area of the intranuclear chromatin excluding the nucleolus, shown as (b), was measured. (C) The area of condensed chromatin, defined as the area with a grayscale value between 193 and 256, was measured (red). The nucleoplasmic chromatin score (Nuc-CS) was defined as (c) / (b) ×100 (%). (D) The area of perinuclear condensed chromatin, defined as (d), and the length of the inner nuclear perimeter were automatically measured (outlined in red in (D)). The perinuclear chromatin score (Per-CS) was defined as (d) / the length of the inner nuclear perimeter (pixel).

Fig 4. Variability in the Nucleoplasmic Chromatin Score (Nuc-CS) and the Perinuclear Chromatin Score (Per-CS).

The variability in Nuc-CS and Per-CS in 6 Group N patients was measured. Six patients (Patients G—L) were randomly selected from Group N. In each patient, Nuc-CS was assessed for all observable nuclei; the variability is shown in the box plot. The top and bottom error bars represent the 90th and 10th percentiles, respectively. The top and bottom tiles of the box represent the 75th and 25th percentiles, respectively. The horizontal bar within the box represents the median.

Fig 5. Relationship between the Nucleoplasmic Chromatin Score (Nuc-CS), Perinuclear Chromatin Score (Per-CS), and Clinical Parameters.

Scatter plots of clinical parameters are shown. % Fib: area of fibrosis within the specimen, LVEF: left ventricular ejection fraction, LVEDD: left ventricular end-diastolic diameter, BNP: brain natriuretic peptide. p: correlation coefficient.

Fig 6. Distribution of Chromatin Score Values (Nuc-CS and Per-CS) and Cardiac Events.

(A) Relationship between Nuc-CS and cardiac events. A white circle represents a patient who did not experience a cardiac event and a dark circle represents a patient who experienced a cardiac event. (B) Relationship between Per-CS and cardiac events. A white circle indicates a patient who did not experience a cardiac event and a dark circle represents a patient who experienced a cardiac event. (C) Receiver operating characteristic (ROC) curve analysis for Nuc-CS in predicting ventricular assist device (VAD) implantation 1 year after endomyocardial biopsy (EMB). (D) ROC curve analysis for Per-CS predicting VAD implantation 1 year after EMB. (E) ROC curve analysis for BNP (black), LVEDD (light blue), LVEF (dark blue), and %Fib (gray) in predicting VAD implantation 1 year after EMB. AUC, area under the curve; BNP, brain natriuretic peptide; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; %Fib, area of fibrosis within a specimen.

Fig 7. Algorithm for DCM Therapy Based on Myocardial Biopsy Parameters.

The first step is morphometric analysis of cardiomyocyte nuclei. When chromatin in the nuclear periphery is discontinuous and there is a homogeneous aggregation of particles in the nucleoplasm, the patient is classified as Group A, which is associated with poor prognosis. When a patient does not have these characteristics, a second step, which involves additional quantitative analysis, is added. Based on the chromatin scores Nuc-CS and Per-CS, a treatment plan can be determined.