Mitochondrial ultrastructure in cardiac myocytes is associated with the cardiovascular death in patients with heart failure

Abstract

BACKGROUND

Mitochondria are considered a promising target for the treatment of cardiovascular disease. However, there has not been a direct association confirmed between mitochondrial ultrastructure in cardiac myocytes and cardiovascular death (CVD) in patients with heart failure (HF).

AIM

To evaluate the association between mitochondrial ultrastructure in cardiac myocytes and CVD in patients with HF who underwent coronary artery bypass grafting (CABG).

METHODS

It was a prospective study of HF patients that used electron microscopy of human cardiac myocytes. A total of 40 patients with HF (left ventricular ejection fraction < 50%) with coronary artery disease who underwent CABG were included (ClinicalTrials.gov: NCT05770349). Right atrial appendage biopsies were obtained during CABG. Ultrastructural analysis of mitochondria was performed using a JEM-1400 transmission electron microscope. The “Total area of interfibrillar mitochondria” and the “Ratio of mitochondrial membrane lengths” were calculated. Patients were followed for 12 months: 6 CVDs were recorded (15%). Statistical analysis was performed using IBM SPSS Statistics version 21; statistical significance was defined as P < 0.05.

RESULTS

From 3 to 5 Longitudinal sections were examined at 5000 × magnification and at 15000 × magnification for each patient (a total of over 250 micrographs were analyzed). The median of the total area of interfibrillar mitochondria was 41% (34; 63); the median of the ratio of the outer membrane length to the inner mitochondrial membrane length was 30% (22; 36). Univariate analysis demonstrated statistically significant differences between the ratio of mitochondrial membrane lengths in patients with and without CVD [29% (20, 33.5) vs 41% (35, 45), P = 0.001]. Patients with CVDs were also more often hospitalized due to HF before CABG and had a long duration of hospitalization after CABG.

CONCLUSION

A disruption in the mitochondrial ultrastructure in cardiomyocytes in the right atrial auricle might be associated with the development of CVD in patients with HF who underwent CABG.

Key Words: Heart failure; Electron microscopy; Mitochondrial ultrastructure; Coronary artery bypass grafting; Cardiovascular death

Core Tip: A prospective observational study using electron microscopy of right atrial appendage cardiomyocytes in patients with heart failure and coronary artery disease who underwent coronary artery bypass grafting was conducted. It was shown that the internal ultrastructure of cardiomyocyte mitochondria, calculated as the ratio of the lengths of the outer and inner membranes, may be associated with the development of fatal outcome during a 12-month prospective follow-up.

INTRODUCTION

The disruption of mitochondrial structure, function, and dynamics is currently recognized as an important mechanism in the development and progression of heart failure (HF)[1]. HF is characterized by the accumulation of fragmented and damaged mitochondria, with disorganization of their internal structures, leading to an inevitable deterioration in bioenergetics in cardiomyocytes, impairing systolic and diastolic heart functions[1-4]. The ultrastructural parameters of mitochondria in cardiomyocytes in HF are related to the severity of HF symptoms, such as limited exercise tolerance, peak oxygen consumption during exercise, left ventricular ejection fraction (LVEF), and the concentration of the N-terminal fragment of pro B-type natriuretic peptide[5,6].

It is only recently that research has been conducted to demonstrate the relationship between mitochondrial structure in cardiomyocytes and the prognosis of patients with HF. For example, a group of Japanese scientists showed that excessive mitochondrial fission is observed in patients with more severe HF, and it is independently associated with adverse outcomes for patients with HF (excessive mitochondrial fission was seen in patients with severe HF status and was associated with adverse outcomes in these patients)[7]. However, this study focuses on the size of mitochondria, rather than their internal structure, which needs further investigation. In another study also conducted in Japan, disorganization of the cristae in cardiomyocyte mitochondria (P = 0.002) was significantly associated with endpoints [composite of cardiovascular death (CVD) and unplanned hospitalization for HF], independent of age (P = 0.115), systolic blood pressure (P = 0.004), B-type natriuretic peptide level (P = 0.042), and mitral regurgitation (P = 0.003)[8]. This is an important result obtained in a Japanese population of patients with non-ischemic cardiomyopathy. However, it is necessary to conduct research on other populations of people as well as on other etiologies of HF.

Thus, the aim of our study was to assess the association between the characteristics of cardiomyocyte mitochondria ultrastructure and the development of fatal outcomes from CVD in patients with HF of ischemic etiology who underwent coronary artery bypass grafting (CABG).

MATERIALS AND METHODS

This single-center prospective study (protocol registered at ClinicalTrials.gov: NCT05770349). The study protocol adhered to the principles of the Declaration of Helsinki (2024) and was approved by the local Ethics Committee of the Cardiology Research Institute, Tomsk National Research Medical Center of the Russian Academy of Sciences (Russia), approval No. 241, dated March 9, 2023.

Study population

We screened patients (n = 851) scheduled for CABG for eligibility and exclusion criteria between May 2023 and May 2025. Inclusion criteria included the presence of HF with LVEF < 50%, atherosclerotic plaques of 70% or more in at least two major coronary arteries, a decision by the cardiology team to perform on-pump CABG, signed informed consent, and availability of biomaterial samples. Exclusion criteria included refusal of revascularization or participation, need for additional cardiac surgical interventions other than CABG, active oncological diseases, presence of implanted devices, severe renal dysfunction (estimated glomerular filtration rate by the chronic kidney disease epidemiology collaboration equation < 30 mL/minute/1.73 m²), infiltrative heart diseases, acute infections or exacerbation of chronic somatic diseases, severe chronic obstructive pulmonary disease, bronchial asthma, type 1 or 2 diabetes mellitus, and anemia.

Participant recruitment took place at a single center between May 2023 and May 2024. All 552 patients scheduled for CABG were screened for inclusion and exclusion criteria. As a result, 40 patients were enrolled in the study and underwent CABG. The follow-up period for patients was 12 months. We had no exclusion criteria during follow-up. After 12 months, an in-person visit was conducted, and if this was not possible, a telephone contact was made with the patient or their relatives in the event of the patient's death. The primary endpoint in the study was the combined primary endpoint (calculated as a percentage) of patients with one or several outcomes, including CVD and/or hospitalization for decompensated HF, or the need for intravenous administration of diuretics and/or acute ischemic events, or repeated unplanned revascularization, or unplanned implantation of cardiac implantable devices. CVD was assessed as a secondary endpoint. We classified CVD into the following categories: Acute myocardial infarction, sudden cardiac death, HF, stroke, cardiovascular procedure, cardiovascular hemorrhage, and other cardiovascular causes[9]. The flow diagram for the study design is presented in Figure 1.

Figure 1 Flow diagram for the study design. CABG: Coronary artery bypass grafting.

The gathered data encompassed patient-reported symptoms, medical history, routine laboratory and instrumental examinations of all patients, including echocardiography and measurement of N-terminal fragment of pro B-type natriuretic peptide. During coronary artery bypass surgery, biopsy samples were obtained from the right atrial appendage. The samples were examined using a JEM-1400 transmission electron microscope (JEOL Ltd., Japan). The images were processed using ImageJ software[10].

In the micrographs taken at a magnification of 5000 times (Figure 2), the “Total area of interfibrillar mitochondria” was determined by calculating the ratio of the total area occupied by all the mitochondria situated between the contractile fibers of the cardiomyocytes and the total area in the interfibrillar space. Measurements were performed on three micrographs per patient to calculate the arithmetic mean, with values expressed as percentages[10]. This parameter reflects the density of mitochondrial distribution between the myofibrils of the cardiomyocyte.

Figure 2 Micrograph of a cardiomyocyte from the auricle of the right atrium of a patient with ischemic heart failure (magnification × 5000). An example of delineation of mitochondria and interfibrillary space is shown (yellow line), which is used to calculate the “Total area of the interfibrillar mitochondria” indicator. A: Initial view; B: Yellow lines indicate the contours of mitochondria and the contour of the interfibrillar space.

On the micrographs at × 15000 magnification (Figure 3), the ratio of the outer mitochondrial membrane length to the inner mitochondrial membrane length was determined (“Ratio of mitochondrial membrane lengths”). This was done for three micrographs for each patient. For each micrograph, this parameter was calculated for three mitochondria. The result was calculated as an average, and the value was expressed as a percentage. This parameter reflects alterations in the internal mitochondrial structure, specifically a reduction in the number of cristae.

Figure 3 Micrograph of a cardiomyocyte from the auricle of the right atrium in a patient with ischemic heart failure (magnification × 15000). An example of the separation of the inner and outer mitochondrial membranes is shown in the micrograph (yellow line), which can be used to calculate the ratio of the outer to inner mitochondrial membrane length. A: A yellow line outlines the outer membrane of a single mitochondrion; B: Yellow lines indicate the outer and inner membranes of three mitochondria.

Then, a comprehensive index was determined, encompassing both the ultrastructural features of mitochondria (taking into account their opposing orientations) using the formula: “Total area of interfibrillar mitochondria”/“Ratio of mitochondrial membrane lengths”. This parameter reflects the combination of mitochondrial characteristics, specifically both the density of their distribution between the myofibrils and the alterations in their internal structure.

Statistical analysis of the results was performed using IBM SPSS 21.0 software. Quantitative continuous variables were presented as the median and interquartile interval: M (Q1; Q3). Categorical data was presented as absolute and relative frequencies, n (%). Continuous variables in independent samples were analyzed using the Mann-Whitney test. To determine the connection between the development of a fatal outcome during prospective observation and the values of quantitative mitochondrial ultrastructure parameters, receiver operating characteristic (ROC) curve analysis was used. Univariate logistic regression was performed to assess the independent influence of factors. Differences were considered statistically significant at P ≤ 0.05.

RESULTS

Clinical and anamnestic characteristics of patients

The study group consisted predominantly of men (87.5%) with a median age of 66 (57; 71) years. More than half of the patients had a positive family history of cardiovascular disease, 97.5% suffered from hypertension, 25.0% of patients had been hospitalized for HF within 12 months prior to study inclusion, and 85% of patients had a history of myocardial infarction. Detailed characteristics of the patients are presented in Table 1.

Table 1 Clinical and anamnestic characteristics of patients included in the study, n (%).

Parameter		Value
Age, years		66 (57; 71)
Male gender		35 (87.5)
Female gender		5 (12.5)
Arterial hypertension		39 (97.5)
A history of stroke		2 (5)
History of myocardial infarction		34 (85)
AF		14 (35)
BMI, kg/m2		30 (26; 33)
NYHA class	I	2 (5)
	II	22 (55)
	III	15 (37.5)
	IV	1 (2.5)
Decompensation of HF in the last 12 months		11 (27.5)
LVEF, %		38 (30; 45.7)
NTproBNP, pg/mL		170 (73; 316)
The result of the 6MWT, m		355 (295; 400)
CCS angina class	I	6 (15)
	II	14 (35)
	III	20 (50)
	IV	0

AF: Atrial fibrillation; BMI: Body mass index; HF: Heart failure; NYHA: The New York Heart Association; LVEF: Left ventricular ejection fraction; NTproBNP: N-terminal fragment pro B - type natriuretic peptide; 6MWT: 6-minute walk test; CCS: Canadian Cardiovascular Society.

Incidence of adverse cardiovascular events

Adverse cardiovascular events were recorded in patients if they occurred within 12 months after CABG. All deaths in the study cohort occurred due to cardiovascular causes. The incidence of major events is presented in Table 2.

Table 2 Incidence of adverse cardiovascular events within 12 months after coronary artery bypass grafting.

Event	Absolute quantity (n)	Relative quantity (%)
Death from cardiovascular diseases	6	15
Hospitalization due to heart failure	6	15
Acute ischemic events requiring unplanned revascularization (myocardial infarction, progressive angina)	1	2.5
Stroke	0
A combined primary endpoint that includes death from cardiovascular causes, hospitalization for heart failure, acute ischemic events requiring unplanned revascularization (myocardial infarction, progressive angina), and acute cerebrovascular accidents	13	32.5

To search for associations between ultrastructural characteristics of mitochondria and development of adverse cardiovascular events, a comparative analysis was performed on three calculated ultrastructural characteristics of these organelles and analyzed endpoints. The results are presented in Table 3.

Table 3 Results of the analysis of associations between the ultrastructural characteristics of mitochondria and the development of adverse cardiovascular events.

Parameter		Total area of interfibrillar mitochondria		The ratio of the length of the outer membrane to the length of the inner membrane		Integral indicator
The end point		Value	P value	Value	P value	Value	P value
Death from CVD	No	41 (35; 50)	0.343	29 (20; 33.5)	0.001	1.5 (1; 2.4)	0.024
	Yes	34 (26; 53)		41 (35; 45)		1 (0.7; 1.4)
Hospitalization due to HF	No	40 (34; 48)	0.299	30.5 (24; 37)	0.556	1.3 (0.9; 2.4)	0.619
	Yes	49 (36; 52)		26 (20; 31)		1.8 (1.3; 2.5)
Progression of HF symptoms	No	37 (32; 47)	0.315	28 (19; 36)	0.712	1.4 (0.9; 2.4)	0.573
	Yes	44 (41; 51)		30 (22; 33)		1.7 (0.6; 1.9)
Combined primary end point (MACE)	No	40 (34; 47)	0.795	28 (19; 36)	0.127	1.4 (0.9; 2.4)	0.279
	Yes	44 (31; 51)		33 (26; 41)		1.4 (0.7; 1.9)

CVD: Cardiovascular diseases; HF: Heart failure; MACE: Major adverse cardiovascular events - serious adverse cardiovascular events.

Thus, the value of the calculated indicators of cardiomyocyte mitochondrial ultrastructure did not differ statistically significantly between patients with and without the registration of a primary endpoint (P > 0.05). However, the indicator of mitochondrial internal structure - the ratio of the outer membrane length to the inner membrane length - was statistically significantly associated with the development of a fatal outcome during a one-year follow-up after CABG. The integral index, reflecting both ultrastructural features, also differed significantly between surviving and deceased patients. Given that the total area of interfibrillar mitochondria was similar in these groups, differences in the integral index were primarily due to mitochondrial internal structural abnormalities.

In order to confirm the independent prognostic significance of the ratio of mitochondrial membrane lengths, a comparative analysis of baseline clinical, anamnestic, and laboratory-instrumental characteristics was conducted between the groups of patients who survived (n = 34) and died (n = 6) during the subsequent one-year prospective follow-up. We aimed to identify potentially influencing factors that differed between the analyzed groups. The results are presented in Table 4.

Table 4 Results of a comparative analysis of the baseline clinical, anamnestic, and laboratory-instrumental characteristics between the groups of patients who survived (n = 34) and died (n = 6) during the subsequent one-year prospective follow-up, n (%).

Parameter	Group 1 (n = 34)	Group 2 (n = 6)	P value
Age, years	65 (56; 69)	71 (58; 72)	0.098
Gender: Male	29 (85.3)	6 (100)	0.423
Atrial fibrillation	9 (26.5)	3 (50)	0.243
History of myocardial infarction	28 (82.4)	6 (100)	0.264
CCS angina class			0.402
I	5 (14.7)	1 (16.7)
II	13 (38.2)	1 (16.7)
III	16 (47.1)	4 (66.6)
NYHA class			0.392
I	1 (2.9)	1 (16.7)
II	20 (58.8)	2 (33.3)
III	12 (35.3)	3 (50)
IV	1 (2.9)	0
Decompensation of HF over the past year	7 (20.6)	4 (66.7)	0.039
Chronic obstructive pulmonary disease	9 (26.5)	2 (33.3)	0.536
A history of stroke	2 (5.9)	0	0.719
Body mass index, kg/m2	30 (27; 34)	28 (23; 30)	0.149
Systolic blood pressure, mm Hg	130 (123; 140)	135 (120; 141)	0.726
Diastolic blood pressure, mm Hg	80 (80; 82)	80 (77.5; 82)	0.999
Heart rate, bpm	72 (62; 78)	72 (68; 86)	0.383
Hemoglobin, g/dL	150 (141; 158)	148 (137; 158)	0.926
Creatinine, mg/dL	94 (76; 103)	100 (88; 109)	0.288
eGFR, mL/minute/m2	75 (62; 88)	70 (60; 76)	0.324
Fasting plasma glucose, mmol/L	6.1 (5.2; 6.3)	5.5 (5; 6.8)	0.592
Total cholesterol, mmol/L	4 (3.1; 4.9)	3.7 (2.8; 4.5)	0.592
Triglycerides, mmol/L	1.66 (1.2; 2.2)	1.44 (1; 1.9)	0.541
Low-density lipoproteins, mmol/L	2.2 (1.4; 3)	2.1 (1; 2.7)	0.425
High-density lipoproteins, mmol/L	0.98 (0.8; 1.1)	1 (0.8; 1.2)	0.582
NTproBNP, pg/mL	163 (73; 326)	170 (74; 349)	0.914
LA, mm	43.5 (40; 47)	43 (39; 56)	0.775
LVEDV, mL	176 (130; 206)	184 (150; 188)	0.999
LVESV, mL	111 (73; 140)	122 (89; 134)	0.835
E/e’	11 (8.8; 15)	13 (9; 17)	0.300
LVMI, g/m2	119 (104; 150)	114 (107; 122)	0.483
sPAP, mm Hg	32 (28; 42)	40 (34; 48)	0.103
LVEF, %	39.5 (30; 46)	35 (28; 41)	0.425
SYNTAX score, points	24 (18; 29)	22 (17; 28)	0.449
Number of shunts	3 (3; 3.25)	3 (2; 3.5)	0.823
Complications	18 (52.9)	6 (100)	0.064
Infectious complications	10 (31.3)	2 (40)	0.999
Duration of hospitalization, bed-days	21.5 (19; 27)	27 (23; 34)	0.043
Drug therapy after discharge
Beta-blockers	28 (82.4)	4 (66.6)	0.740
ACE inhibitor	15 (44.1)	2 (33.3)	0.623
ARBs	5 (14.7)	2 (33.3)	0.600
ARNI	10 (29.4)	2 (33.3)	0.848
Any RAAS inhibitor	30 (88)	6 (100)	0.883
iNGLT2	23 (67.6)	3 (50)	0.711
MRAs	25 (73.5)	3 (50)	0.499
Loop diuretics	25 (73.5)	4 (66.6)	0.882
Iron preparations	13 (38.2)	2 (33.3)	0.820

HF: Heart failure; NYHA: The New York Heart Association; CCS: Canadian Cardiovascular Society; eGFR: Estimated glomerular filtration rate; NT-proBNP: N-terminal fragment of pro B-type natriuretic peptide; LA: Left atrium; LVEDV: Left ventricular end-diastolic volume; LVESV: Left ventricular endsystolic volume; E/e’: Ratio of the maximum rate of early diastolic filling of the mitral flow to the early diastolic rate of movement of the fibrous ring of the mitral valve; LVMI: Left ventricular mass index; sPAP: Systolic pulmonary artery pressure; LVEF: Left ventricular ejection fraction; SYNTAX: Synergy between Percutaneous Coronary Intervention with TAXUS and Cardiac Surgery; ACE: Angiotensin converting enzyme; ARBs: Angiotensin receptor blockers; ARNI: Angiotensin receptors and non-lysine inhibitor; RAAS: Renin-angiotensin-aldosterone system; iNGLT2: Type 2 sodium-glucose co-transporter inhibitor; MRAs: Mineralocorticoid receptor antagonists.

Thus, the cohorts of survivors and deceased patients differed statistically significantly in terms of the duration of hospitalization, which was longer in patients who subsequently developed a lethal outcome. This is probably due to the higher frequency of postoperative complications, although the differences in complication frequency did not reach statistical significance. In addition, there were significant differences in hospitalizations due to decompensation of HF within one year before CABG between the two groups.

The next step was univariate logistic regression analysis. Considering low number of events (6 CVDs), the study cannot utilize the multivariable regression analysis. The mitochondrial internal ultrastructure may be associated with mortality during a one-year follow-up period. The results are presented in Table 5.

Table 5 Results of the univariate logistic regression analysis.

Parameter	B (regression coefficient)	The Wald significance test	P value	OR	95%CI
					The lower limit	The upper limit
Ratio of mitochondrial membrane lengths	0.134	4.440	0.035	1.143	1.009	1.295

OR: Odds ratio; CI: Confidence intervals.

An ROC-analysis was performed to assess the prognostic significance of the ratio of the length of the outer and the inner mitochondrial membranes in relation to risk of death during one-year follow-up after CABG. The area under the curve was 0.907; P = 0.002. The results of ROC-analysis are shown in Figure 4.

Figure 4 Receiver operating characteristic curve of the “Ratio of mitochondrial membrane lengths” for predicting one-year mortality risk after coronary artery bypass grafting. Area under the curve = 0.907, 95% confidence interval: 0.808-0.999, P = 0.002. ROC: Receiver operating characteristic.

The cut-off point for the indicator of the ratio of membranes lengths in predicting a lethal outcome was a value of 34%, with a sensitivity of 100% and specificity of 77%.

A figure with biopsy data from survived and deceased patients demonstrating the differences in mitochondrial structure we provide as a visual illustration of the differences between the groups (Figure 5).

Figure 5 Micrographs of a cardiomyocyte from the auricle of the right atrium in two patients with ischemic heart failure (magnification × 15000). Yellow lines indicate the outer and inner membranes of three mitochondria for each patient to calculate the “Ratio of mitochondrial membrane lengths”. A: Micrograph of the myocardium of a surviving patient, where the “Ratio of mitochondrial membrane lengths” was 21%; B: Micrograph of the myocardium of a deceased patient, where the “Ratio of mitochondrial membrane lengths” was 53% (a predictor of mortality).

DISCUSSION

We present the results of a small study with a complex design, which included a study of the structure of cardiomyocyte mitochondria in patients with HF and coronary artery disease who underwent CABG with artificial circulation. The included patients were mostly male, with a median age of 66 (57; 71) years and a history of cardiovascular disease (85% had a history of myocardial infarction), and all patients had reduced LVEF (< 50%). Almost a third of the patients had experienced HF hospitalization within 12 months before inclusion in the study.

The incidence of the primary composite endpoint, which included death from cardiovascular causes, hospitalization for HF, acute ischemic events requiring unplanned revascularization (myocardial infarction, progressive angina), and acute cerebrovascular events, was 32.5% in this cohort. Almost half of these events were CVDs (15%), which is consistent with the Surgical Treatment for Ischemic Heart Failure study. The incidence of a composite endpoint of death or HF hospitalization during the year of follow-up was 30%-35%, depending on age. The mortality charts presented show that the incidence of death is approximately 10%-15%[11]. Thus, our results are consistent with those of the Surgical Treatment for Ischemic Heart Failure, although the medical treatment of patients has changed significantly in recent years.

Mitochondrial dysfunction plays an important role in the pathogenesis of HF progression, especially considering its ischemic etiology in our study[1]. Unlike previous studies[6-8], which focused on left ventricular cardiomyocytes, we analyzed mitochondria of cardiomyocytes from the right atrial appendage[12]. To date, there is no convincing evidence of the comparability of processes occurring in the mitochondria of left ventricular and atrial cardiomyocytes. However, there is evidence of universality of basic functions of mitochondria in all organs and tissues, including development of pathological processes such as HF. This suggests a systemic negative impact on cardiomyocytes regardless of their location. In previous studies, using light microscopy, it was shown that changes in the myocardium of the right atrial appendage reflect general processes of morphological changes in the heart’s cardiomyocytes and correlate with the prognosis of ischemic cardiomyopathy[13]. Moreover, given that 85% of patients in our cohort had a history of myocardial infarction, biopsy of the left ventricular wall was associated with a high probability of detecting heterogeneity in ultrastructural changes in mitochondria, depending on the location of cardiomyocytes in relation to post-infarction scars.

Despite the study of cardiomyocytes from the right atrial appendage, rather than the left ventricle, our analysis showed a statistically significant association between ultrastructural parameters of the mitochondria of the cardiomyocytes of the right atrial appendage and the development of fatal outcomes during one-year follow-up after CABG. The results of our previous electron microscopy study have demonstrated that the “Total area of interfibrillar mitochondria” was associated with general functional status according to the 6-minute walk (r = 0.395, P = 0.012) and cardiopulmonary exercise tests (r = 0.483, P = 0.002). The “Ratio of mitochondrial membrane lengths” was inversely correlated with LVEF (r = -0.593, P = 0.033)[5]. No statistically significant correlations were found between the analyzed mitochondrial ultrastructure parameters and The New York Heart Association functional class of HF, as well as the characteristics of coronary artery disease. This association with functional parameters in HF may be one explanation for the proposed association of mitochondrial internal ultrastructure with the development of HF death in the study group obtained in the present study. Numerous previous clinical studies confirm the existence of a relationship between functional parameters, such as the results of cardiopulmonary exercise testing[14,15] and the distance of the 6-minute walk tests[16] with prognosis in HF. The relationship between reduced LVEF and prognosis in patients with HF who underwent CABG was convincingly demonstrated in seminal studies in this cohort of patients[11,17]. It can be speculated that, mechanistically, these functional changes and the reduction in LVEF may partially explain the observed relationships between mitochondrial ultrastructure and patient prognosis in our cohort.

Thus, our results are consistent with the few studies conducted on a HF patients[6-8]. These studies were conducted in parallel with our research, and at the time of initiation of our study, these results were not available in the literature. Our findings significantly contribute to understanding the mechanisms of HF progression in patients with low ejection fractions, as the internal ultrastructure of mitochondria was associated with patient outcomes. We calculate it as the ratio of the length of the outer mitochondrial membrane to the length of the inner membrane. This indicator reflects the destruction of the internal structure of mitochondria, and its value increases as the number of cristae decreases in mitochondria. This result agrees fully with the study conducted by Nakano et al[8], in which crest disorganization was significantly associated with endpoints (a combined endpoint including CVD and unplanned hospitalization for HF) independently of age (P = 0.115), systolic blood pressure (P = 0.004), natriuretic peptide B-type level (P = 0.042), and mitral regurgitation (P = 0.003). However, Nakano et al[8] was conducted on a Japanese population; the patients did not undergo CABG, and the myocardial fragments for the study were taken from the left ventricular wall. Therefore, it can be assumed that mitochondrial cristae disorganization is a universal mechanism for the progression of HF with reduced LVEF and is associated with poor prognosis in these patients, which is confirmed by our results.

Study limitations

The main limitation is the small sample size in our study. However, in the context of studies that analyze electron micrographs of human myocardium compared to clinical, laboratory, and instrumental characteristics, our study is among the largest in terms of the number of patients included and number of micrographs analyzed. Another limitation is that we studied cardiomyocytes from the right atrial appendage rather than the left ventricle itself, but our results are consistent with literature and indirectly confirm the universality of mitochondrial changes in different parts of the heart in HF. Additionally, all patients underwent CABG using cardiopulmonary bypass, which could have affected their prognosis, although the surgical procedures were not different between survivors and decedents in our study.

The evaluation system for mitochondrial structural damage in this manuscript is insufficiently comprehensive, limited to only two ultrastructural parameters: The total area proportion of interfibrillar mitochondria and the ratio of outer mitochondrial membrane length to inner mitochondrial membrane length. It would be highly valuable to evaluate additional quantitative metrics describing mitochondrial morphology and fragmentation, such as the average cross-sectional area of mitochondria and the proportion of fragmented mitochondria and mitochondrial cristae density and the proportion of mitochondria with disorganized cristae. However, we analyzed micrographs without the use of artificial intelligence technologies. The artificial intelligence could have accelerated the process and allowed for analysis of even more mitochondria, which could be a subject for future research.

Our study solely relies on quantitative analysis of transmission electron micrographs for morphological observation. Western blotting analysis would be highly appropriate for molecular validation to detect differential expression of proteins associated with mitochondrial function, mitochondrial injury, and apoptosis. Comprehensively addressing this aspect lies beyond the scope of our current work, but we see it as a crucial next step.

CONCLUSION

Disruption of the internal structure of mitochondria in cardiomyocytes in the right atrial appendage manifests as a decrease in cristae number might be associated with the development of CVD in patients with HF who underwent CABG. The shorter the length of the inner mitochondrial membrane compared to the outer membrane, the worse the prognosis for patients 12 months after CABG.