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Revisiting the pathogenic role of insulin resistance in Duchenne muscular dystrophy cardiomyopathy subphenotypes

AbdelMassih, Antoine Fakhrya,,b; Esmail, Reema; Zekri, Hanana; Kharabish, Ahmedc,,d; ElKhashab, Khalede; Menshawey, Rahmaf; Ismail, Habiba-Allahf; Afdal, Peterf; Farid, Erinig; Affifi, Omneyah

Author Information
Cardiovascular Endocrinology & Metabolism: December 2020 - Volume 9 - Issue 4 - p 165-170
doi: 10.1097/XCE.0000000000000203
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Abstract

Introduction

Duchenne muscular dystrophy (DMD) is an X-linked disease that affects 1 in 3600–6000 live male births [1]. The majority of patients with DMD, after their third decade of age, have established cardiomyopathy. Although clinically overt heart failure may be delayed or absent, due to relative physical inactivity, cardiac disease is a major cause of death in patients with muscular dystrophies [2].

Previous studies point to predominant subepicardial/midmyocardial fibrosis in DMD, suggesting that the subepicardium is the earliest layer that is involved in the disease process. Giglio et al. [3] screened the pattern of late gadolinium enhancement (LGE) in DMD carriers, and found no patients with subendocardial involvement. Other experimental studies point at another type of dysfunction in the subendocardium this has been proven in a limited study in dogs with DMD, and bradykinin has been effective in the reversal of such process [4].

The presence of two possible different forms of myocardium involvement, raise a question about the mechanisms underlying such discrepancy. It is well known that fibrosis starts in the subepicardium and midmyocardium, and that this fibrosis is largely driven by a state of systemic immune-mediated inflammation in patients with DMD. The inflammatory response occurs in response to myofiber damage, and this starts a cascade of inflammation that exacerbates the process of myofiber loss [5].

However, this is not the only mechanism suggested to be involved in myocardial injury in DMD. In 2012, Hahn et al. published a case report of severe proliferative retinopathy in DMD cases. The mechanisms involved in such neovascularization are not well understood. There is evidence of a degenerative paradigm underlying the neovascularization in DMD. Endothelial rarefaction happens the same way as the loss of myofibers, and starts the course of inflammation that ends in neovascularization and vascular occlusion [6]. Another potential mechanism in vascular occlusion might be the state of dyslipidemia commonly developing in DMD [7]. These two mechanisms might be involved in the induction of subendocardial injury in patients with DMD.

The primary outcome parameter in this study is to detect whether two forms of DMD cardiomyopathy exist. The secondary outcome parameter is to relate the possible presence of two patterns to certain biomarkers such as creatinine kinase as a marker of myofiber damage, or lactate dehydrogenase (LDH) as a compound marker of endothelial rarefaction and myofiber loss, or to some metabolic alterations linked to endothelial dysfunction, such as lipid profile or insulin resistance.

Patients and methods

Study population

This study was designed as a case–control, cross-sectional study on pediatric asymptomatic (with no cardiac symptoms) patients with DMD, and was performed between February 2018 and February 2019. The patients were recruited from the neurometabolic clinic of Cairo University Children Hospital, Egypt (a tertiary care center).

Inclusion criteria included DMD diagnosis, age between 8 and 18 years.

Exclusion criteria were any cardiac lesions apart from patent foramen ovale.

Patients were initially subjected to cardiovascular MRI (CMR), performed using a 1.5-T MR system (INTERA, Philips Medical Systems, Best, the Netherland) with a cardiac five-element phased-array receiver coil. All images were acquired with ECG-gating, breath-hold steady-state for functional analysis, and myocardial enhancement by LGE. LGE images were assessed visually and considered positive to a signal intensity threshold of >2 SD above the mean intensity of a remote reference region [18]. According to the LGE location, patients were divided into two groups were as follows:

  • (1) Subendocardial group (SENDO) where LGE is located in the subepicardium.
  • (2) Subepicardial/midmyocardial (SEPMI) where LGE was located in the subendocardium.

CMR examinations were interpreted by two CMR trained physicians.

Study methods

In the SENDO and SEPMI groups, the following were assessed:

  • (1) Body weight and height measurement to calculate the body mass index (BMI).
  • (2) Level of activity was assessed using Ambulation Function Classification system [8].
  • (3) New York Heart Association classification was performed on all patients [9].
  • (4) Echocardiography for left ventricle functions as follows according to the guidelines of the American Society of Echocardiography: [10–12].
    • (a) For diastolic function
      • (i) Conventional Doppler and Tissue Doppler (Tissue doppler imaging) have been used to determine the left ventricle E/E´ ratio: the ratio of early mitral inflow velocity to average early diastolic velocities of the basal septum and mitral annulus.
      • (ii) Digitally stored images were measured offline, left atrial maximal volume was measured using three left atrium dimensions (LADs) as described previously: 13 left atrium volume = π/6 (LAD 1 × LAD 2 × LAD 3) where LAD 1 = LAD by M-mode, LAD 2, and LAD 3 are measurements of short-axis and long-axis LAD in the apical four-chamber view, respectively, using Teo et al. [13] approach just before the mitral valve opening.
    • (b) Real-time 3D Echocardiography: Full-volume acquisition of the left ventricle was obtained by harmonic imaging from the apical approach. All data sets were analyzed off-line using commercially available software (4D AutoLVQ, GE-Vingmed, Horten, Norway). The software automatically identified the left ventricle cavity endocardial border in 3D. The operator performed all the necessary adjustments manually, in order to correctly place the endocardial border. After the adjustments, software provided left ventricular ejection fraction (LVEF).
  • (c) 2D-Speckle tracking myocardial layer strain discriminating echocardiography (MLSD-STE) of the left ventricle [14] by Transthoracic echocardiography was performed using General Electric (Vivid-7/9, Horten, Norway):
    • (i) The endocardial border of the heart was manually traced at end-systole.
    • (ii) The software automatically then detected and calculated for each of the 17 segments the longitudinal strain (shortening) of the subepicardial layer of the myocardium, alternatively called ‘Epicardial Strain’ (Epi-S), and that of subendocardial layer of the myocardium, also known as ‘Endocardial Strain’ (Endo-S), and the myocardial strain.
    • (iii) The average of the 17 segments was then calculated for each of the 3 longitudinal strain values, to calculate the midmyocardial strain alternatively called Global Longitudinal Strain (GLS), and the subepicardial GLS (EPI-LS) and ENDO-LS. In each group of patients, the number and percentage of patients with lower ENDO-LS compared to EPI-LS, and vice versa was determined. Echocardiography was performed by two senior cardiologists who were blinded to clinical data, and to the results of each other, and Kappa analysis was performed to determine the Interobserver variability as well as the level of agreement between cardiac MRI results and MLSD-STE results.
  • (5) Biochemical profile including:
    • (a) Total lipid profile: Low-density lipoproteins (LDL), high-density lipoproteins (HDL), total cholesterol, triglycerides.
    • (b) Fasting Glucose insulin ratio (FGIR) was calculated using the following equation: Fasting insulin (µU/mL) × fasting glucose (mmol/L)/22.5.FGIR was used to determine insulin resistance in each group of patients, It has the advantage of being easily performed, unlike oral glucose tolerance test which depends on serial serum glucose measurements [15].
    • (c) Muscle enzymes: creatinine kinase and LDH. For creatinine kinase and LDH assay, the samples were collected in dry tubes. The serum LDH and creatinine kinase assays were performed with a spectrophotometer at 340 nm. The kit was provided for creatinine kinase by BioAssay Systems, USA with a reference range between 60 and 174 U/L. The kit for LDH was provided by Cypress diagnostics, Belgium with a reference range between 160 and 320 U/L [16,17].

Statistical analysis

Data were statistically described in terms of mean ± SD, and percentages when appropriate. Comparison of numerical variables between the study groups was done using Student’s t-test for independent samples when normally distributed, and Mann–Whitney U test for independent samples when not normally distributed. Scatter plot was performed to represent the relationship between insulin resistance and left ventricle function. Receiver operating characteristic analysis was performed and represented by interactive dot diagram to illustrate the diagnostic accuracy of creatinine kinase/LDH ratio in the differentiation of DMD cardiomyopathy subphenotypes.

Results

Tables 1 and 2 show the demographic and biochemical data between the two study groups. BMI in the SENDO group was found to be slightly higher than the SEPMI group. There were no noticeable differences in the level of disability between the two groups. Cardiac symptoms were not overt in the two studies group, as most of the patients pertained to NYHA class I. Lipid profile did not show any statistically significant difference between the two study groups, except for triglycerides which were slightly elevated in SENDO group than in the SEPMI group. Regarding muscle enzymes, SENDO group displayed more LDH elevation, while SEPMI group showed more evident creatinine kinase elevation in this aspect. Figure 1 which is an interactive dot diagram shows that creatinine kinase/LDH ratio can differentiate between the two myocardial subphenotypes with a sensitivity of 100%.

Table 1 - Demographic and clinical data of the two study groups
SENDO (n = 26) SEPMI (n = 34) P value
Age (years) 9 ± 2 9 ± 1 NS
BMI 24 ± 1 23 ± 1 NS
New York Heart Association Classification class n (%) Class I: 25 (96%) Class I: 32 (94%) NS
Class II: 1 (4%) Class II: 2 (6%) NS
Class III: 0 (0%) Class III: 0 (0%) NS
Class IV: 0 (0%) Class IV: 0 (0%) NS
Ambulatory Classification System for DMD n (%) Level 1: 22 (84%) Level 1: 28 (82%) NS
Level 2: 4 (16%) Level 2: 6 (18%) NS
Level 3: 0 (0%) Level 3: 0 (0%) NS
Level 4: 0 (0%) Level 4: 0 (0%) NS
Level 5: 0 (0%) Level 5: 0 (0%) NS
P < 0.05 was considered statistically significant.
BMI, body mass index; DMD, Duchenne muscular dystrophy; n, number; NS, nonsignificant; SENDO, subendocardial group of DMD patients; SEPMI, subepicardial-midmyocardial group of DMD patients.

Table 2 - Biochemical data of the two study groups
SENDO (n = 26) SEPMI (n = 34) P value
HDL (mg/dL) 47 ± 33 57 ± 15 NS
LDL (mg/dL) 151 ± 58 147 ± 11 NS
Triglycerides (mg/dL) 91 ± 33 71 ± 19 0.04
Total cholesterol (mg/dL) 151 ± 58 147 ± 11 NS
FGIR (ratio) 7 ± 1 5 ± 1 <0.001
LDH (U/L) 570 ± 76 181 ± 61 <0.001
Creatinine kinase (U/L) 570 ± 22 1409 ± 19 <0.001
FGIR, fasting glucose-insulin ratio; HDL, high-density lipoproteins; LDH, lactate dehydrogenase.

Fig. 1
Fig. 1:
Interactive dot diagram showing the ability of creatinine kinase/LDH ratio to discriminate between subendocardial and subepicardial phenotypes of DMD. DMD, Duchenne muscular dystrophy; LDH, lactate dehydrogenase; Sens, sensitivity; Spec, Specificity.

FGIR was more elevated in SENDO group, denoting higher insulin resistance.

Regarding echocardiographic data in Table 3, left ventricle systolic functions as expressed by 3D derived LVEF was more altered in the SENDO GROUP than the SEPMI group, without achieving enough statistical significance.

Table 3 - Echocardiographic and CMR data of the two study groups
SENDO (n = 26) SEPMI (n = 34) P value
LVEF (%) 59 ± 12 61 ± 10 NS
LV E/E´ (ratio) 9 ± 3 7 ± 2 0.04
E’: Mitral annular early diastolic velocity 8.2 ± 2.1 9.8 ± 1.5 0.05
Septal E’: Basal septal early diastolic velocity 7.3 ± 1.1 10 ± 2 0.03
LV GLS (%) 14 ± 1.3 14.1 ± 1.2 NS
Indexed left atrium volume maximum 22 ± 3 17 ± 2 0.02
Left ventricle subepicardial strain (%) 17.6 ± 2 11.8 ± 1 <0.001
Left ventricle subendocardial strain (%) 10 ± 0.9 16.4 ± 1.9 <0.001
Patients with subendocardial strain>subepicardial strain (n/%) 8 (31%) 30 (88%) <0.001
Patients with subendocardial strain<subepicardial strain (n/%) 18 (69%) 4 (12%) <0.001
LVEDVI by CMR (mL/m2) 76 ± 12 77±11 NS
LVEF by CMR (mL/m2) 61 ± 12 62 ± 14 NS
CMR, cardiovascular MRI; EDVI, end-diastolic volume index; EF, ejection fraction; GLS, global longitudinal strain; LV E/E´, the ratio of early mitral inflow velocity to average early diastolic velocities of the basal septum and mitral annulus.

Similarly, left ventricle E/E´ ratio and left atrial maximal volume as surrogate markers for left ventricle diastolic functions were higher in SENDO group denoting more diastolic dysfunction.

Figure 2 is a regression curve showing the relationship between subendocardial involvement and insulin resistance as expressed by FGIR. In this aspect, FGIR was higher implying more insulin resistance in SENDO group (Table 1).

Fig. 2
Fig. 2:
Scatter plot (regression curve) showing relationship between FGIR and subendocardial GLS. FGIR, fasting glucose-insulin ratio; GLS, global longitudinal strain; r, correlation coefficient; P, Pearson coefficient for statistical significance.

Level of agreement between cardiac MRI and MLSD-STE was high with a Kappa coefficient of 0.82.

Kappa coefficient between the two physicians interpreting the CMR data was 0.91.

Kappa coefficient between the two physicians interpreting the Echocardiography examinations was 0.85.

Discussion

DMD is a fatal myopathy with expected cardiorespiratory failure between the second decade and third decade. Left ventricular mechanics and pathogenesis of its involvement have long been explained by the predominance of fibrosis involving the subepicardium earlier than the subendocardial layer of the myocardium [4].

Moreover, there is a current wide consensus that DMD is not only a disease related to muscles, but that its systemic effects extend to cause metabolic alterations. These metabolic alterations, including dyslipidemia and insulin resistance with subsequent defective glucose transport, do not seem to be triggered by the state of stagnation and obesity related to disability; yet there appear to be molecular mechanisms triggering such alterations [18]. Another finding might confirm such hypothesis, which is the absence of statistically significant difference between both groups, as regards the level of disability and subsequently at the level of physical activity.

Despite this wide consensus, to our knowledge, no studies have focused on the relationship of these metabolic alterations and the state of myocardial injury in patients with DMD.

In our series, we used LGE to discriminate two DMD groups with discrepant forms of myocardial injury; a group with predominant subepicardial/midmyocardial dysfunction (SEPMI) and a group with predominant subendocardial involvement (SENDO). Comparing the metabolic alterations across the two groups revealed the predominance of insulin resistance in SENDO group which was statistically significant. This was not justified by a significant difference in BMI, or in the level of physical activity as assessed by Ambulation Function Classification system. This might indicate a hereditary predisposition to insulin resistance in certain subgroups of patients with DMD. This contradicts the false fixed belief that insulin resistance in DMD is induced by obesity and physical inactivity [19]. The association of subendocardial involvement and insulin resistance was found in our previous series in patients with CKD; insulin resistance might be implicated in the pathogenesis of arteriosclerosis, with subsequent impairment of blood flow to the myocardium, especially the subendocardial layer being the most vulnerable in cases with arteriosclerosis [20,21].

It is well known that muscle enzymes are elevated in DMD due to muscle wear and tear, creatinine kinase is the chief marker of muscle damage and is rarely confounded by other tissues’ involvement, while LDH is involved in many other cellular processes. Nakayama et al. reported in his series that pulmonary thrombosis is not an uncommon finding in the context of DMD. The pathogenesis of hypercoagulable state as suggested by Pritchard et al. [22]. involves consumption of naturally occurring vasodilators due to myofiber necrosis, and intravascular hemolysis due to fragile RBCs cytoskeleton. There is evidence that in DMD, LDH levels rise to a greater extent during episodes of thrombosis, and this is in line with what similarly occurs in sickle cell disease, where intravascular hemolysis depletes circulating nitric oxide and correlates well with serum LDH levels [23,24,25].

In our series, LDH was more elevated in SENDO group than SEPMI group, and the reverse occurs regarding creatinine kinase levels. Creatinine kinase/LDH ratio might serve as a discrimination marker between both cardiac phenotypes of the disease with a sensitivity of 100%.

Transmural strain or MLSD-STE, has been first unleashed in 2009 by Adamu et al., since then there has been scarce data about its role in the pediatric age group. The first study to our knowledge to implement it in pediatrics was by our working group, to detect the differences in myocardial involvement between different hemoglobinopathies. This study is by the far the first to compare the potential agreement between CMR and this experimental emerging technique in the differentiation of myocardial layer involvement. There seems that a wide range of agreement between the two methods, subendocardial function was more reduced in 69% of the SENDO group, and subepicardial function was reduced to a greater extent in 88% of patients in the SEPMI group.

Finally, Diastolic and Systolic functions of the left ventricle seem to be more deeply involved in the SENDO group. This finding is in agreement with previous studies showing that the subendocardium impacts myocardial functions, especially diastolic functions, to a greater extent than the subepicardium [26].

Conclusion and limitations

This study points at the presence of different cardiac phenotypes in DMD. These cardiac phenotypes might be induced by metabolic alterations which are a consequence of DMD disease process. DMD seems to be a more complex systemic disease, rather than a simple muscular dystrophy. More studies need to be performed at a molecular level to elucidate the potential hypotheses suggested by our study. Two major limitations to the study are the absence of phenotype-genetic correlation to assess if genetic variabilities are related to the observed discrepant cardiac phenotypes, and the relatively small sample size.

Acknowledgements

We want to thank every student enrolled with us in the research accessibility team, these young promising students and interns are doing a very faithful job. Their contribution and their efforts are very palpable and we are proud as working group of professors to give them this chance and to witness the pivoting change it brings to the workflow in addition to the tremendous hope it adds to our lives.

Conflicts of interest

There are no conflicts of interest.

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