Background
Methods
Patients
Assessment of skeletal muscle proteolysis
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We measured urinary 3-methylhistidine (3-MH) to assess skeletal muscle proteolysis. The amino acid, 3-MH, is generated by post-translational methylation of histidine residues derived from actin and myosin degradation [12]. It cannot be further catabolized, or recycled for protein synthesis, and is excreted in the urine. Therefore, it reflects the myofibril protein turnover [13]. All patients collected 24-h urine into bottles containing hydrochloric acid. The 3-MH in the urine was measured by a precolumn derivatization using reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDSTAG WAKO, FUJIFILM WAKO Inc. Osaka, Japan), followed by reversed-phase high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS-2020, Shimadzu Corporation, Kyoto, Japan). The urine creatinine was analyzed by a Jaffe reaction. The amount of skeletal muscle proteolysis was determined as the ratio of the urinary 3-MH to creatinine (3-MH/Cr) because 3-MH is influenced by the renal function. All patients were asked to refrain from ingesting soy-based products and meat 3 days before the measurement because of a possible effect on the 3-MH.
Assessment of lower limb muscle mass and strength
Six-minute walk test
Blood samples and echocardiography
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Blood tests and two-dimensional echocardiography were performed upon study enrolment. To avoid the effect of exercise testing on the IL-6, blood sampling was performed following echocardiography in the supine position. Blood samples were collected from a peripheral vein into tubes containing aprotinin and ethylene diamine tetra acetic acid. The blood samples were centrifuged at 1000×g at 4 °C for 15 min to isolate serum or plasma and store it at − 80 °C until assayed. The plasma BNP concentration was measured using a chemiluminescence enzyme immunoassay for human BNP (LLMIPULSE Presto, Fujirebio Inc. Tokyo, Japan). The serum IL-6 concentration was measured using a chemiluminescence enzyme immunoassay for human IL-6 (human IL-6 measurement kit, Fujirebio Inc. Tokyo Japan) and serum high-sensitivity C-reactive protein (CRP) levels via a latex-enhanced hs-CRP immunoassay (N-Latex CRP II, Siemens Healthineers, Tokyo, Japan). A single echocardiographer who was blinded to the patients’ clinical information performed offline echocardiographic analysis using a Vivid 7 (GE Healthcare, Boston, Massachusetts, USA).
End point and follow-up
Statistical analysis
Results
Clinical characteristics of the patients
Characteristic | Control (n = 83) | Heart failure (n = 78) | P-value |
---|---|---|---|
Age (years) | 72 ± 5 | 73 ± 8 | 0.21 |
Female | 24 (29) | 28 (36) | 0.15 |
Body mass index (kg/m2) | 23.3 ± 3.5 | 22.8 ± 4.2 | 0.42 |
Systolic blood pressure (mmHg) | 123 ± 17 | 125 ± 23 | 0.69 |
Diastolic blood pressure (mmHg) | 75 ± 13 | 76 ± 14 | 0.51 |
Cardiac rhythm | 0.40 | ||
Sinus rhythm | 35 (42) | 23 (29) | |
Paroxysmal/persistent AF | 48 (58) | 55 (71) | |
Medical comorbidities | |||
Coronary artery disease | 6 (7) | 6 (8) | 0.86 |
Valvular disease | 1 (1) | 10 (13) | < 0.001 |
Hypertension | 30 (36) | 38 (49) | 0.11 |
Diabetes | 10 (12) | 16 (21) | 0.14 |
Dyslipidemia | 17 (20) | 14 (18) | 0.68 |
TIA/Stroke | 4 (5) | 11 (14) | < 0.05 |
Laboratory data | |||
Hemoglobin (g/dL) | 13.8 ± 11.7 | 12.7 ± 2.0 | < 0.001 |
Creatinine (mg/dL) | 0.83 ± 0.19 | 0.88 ± 0.39 | 0.36 |
eGFR (mL/min/1.73m2) | 68 ± 17 | 66 ± 18 | 0.39 |
CRP (mg/dL) | 0.16 ± 0.26 | 0.70 ± 1.16 | < 0.001 |
IL-6 (pg/mL) | 3.30 ± 3.71 | 8.08 ± 7.05 | < 0.001 |
Creatine kinase (IU/L) | 91 ± 33 | 92 ± 50 | 0.87 |
BNP (pg/mL) | 41 ± 28 | 328 ± 249 | < 0.001 |
LVEF (%) | 56 ± 11 | 45 ± 16 | < 0.001 |
LAD (mm) | 35 ± 5.8 | 40 ± 7.4 | < 0.001 |
Medications | |||
β-Blocker | 59 (71) | 75 (96) | < 0.001 |
ACE-I/ARB | 29 (35) | 66 (85) | < 0.001 |
Loop diuretics | 4 (5) | 31 (40) | < 0.001 |
Aldosterone blocker | 5 (6) | 18 (23) | < 0.01 |
Calcium blocker | 23 (28) | 19 (24) | 0.56 |
Statin | 16 (19) | 27 (35) | < 0.05 |
Aspirin | 10 (12) | 21 (27) | 0.16 |
Anticoagulant | 48 (58) | 56 (72) | 0.09 |
Lower limb skeletal muscle measurements and the 6-min walk test
Control (n = 83) | Heart failure (n = 78) | P-value | |
---|---|---|---|
Thigh circumference (cm) | 48 ± 7.2 | 42 ± 8.2 | < 0.001 |
Thigh muscle thickness (cm) | 2.9 ± 0.2 | 1.8 ± 0.3 | < 0.001 |
Knee extensor strength (kg) | 19.7 ± 6.4 | 13.9 ± 4.4 | < 0.001 |
3-MH/Cr (nmol/g Cr) | 191 ± 144 | 386 ± 445 | < 0.001 |
6-min walk distance (m) | 570 ± 45 | 534 ± 45 | < 0.001 |
Spearman’s rank correlation analyses
Age | Female | AF | CRP | IL-6 | BNP | LAD | LVEF | Thigh circumference | Rectus femoris thickness | Knee extensor strength | 3-MH/Cr | |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Female | − 0.03 | |||||||||||
AF | −0.14 | − 0.04 | ||||||||||
CRP | −0.06 | − 0.16 | − 0.12 | |||||||||
IL-6 | 0.08 | −0.13 | − 0.18 | 0.53§ | ||||||||
BNP | 0.02 | 0.21 | 0.13 | 0.07 | 0.17 | |||||||
LAD | −0.05 | − 0.10 | 0.16 | 0.26* | 0.27* | 0.43§ | ||||||
LVEF | −0.03 | 0.05 | 0.01 | −0.22* | − 0.26* | − 0.15 | − 0.19 | |||||
Thigh circumference | −0.29* | − 0.40# | − 0.15 | −0.15 | − 0.03 | − 0.40# | − 0.17 | −0.02 | ||||
Rectus femoris thickness | −0.32* | −0.40# | − 0.11 | −0.12 | − 0.10 | −0.16 | − 0.02 | 0.09 | 0.58§ | |||
Knee extensor strength | −0.37# | −0.32# | − 0.10 | −0.10 | − 0.15 | −0.31 | − 0.04 | 0.16 | 0.43# | 0.64 § | ||
3-MH/Cr | 0.22* | 0.16 | −0.17 | 0.09 | 0.13 | 0.17 | 0.03 | −0.16 | 0.05 | −0.14 | −0.23* | |
6-min walk distance | −0.29* | −0.14 | − 0.07 | −0.30# | − 0.57§ | −0.41# | − 0.43§ | 0.18 | 0.34# | 0.29* | 0.25* | −0.22* |
Age | Female | AF | CRP | IL-6 | BNP | LAD | LVEF | Thigh circumference | Rectus femoris thickness | Knee extensor strength | 3-MH/Cr | |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Female | −0.05 | |||||||||||
AF | −0.13 | 0.13 | ||||||||||
CRP | 0.13 | 0.10 | 0.11 | |||||||||
IL-6 | 0.22 | 0.07 | 0.21 | 0.51§ | ||||||||
BNP | 0.31# | 0.04 | 0.31# | 0.31# | 0.50§ | |||||||
LAD | 0.05 | −0.07 | 0.22* | 0.16 | 0.05 | 0.24* | ||||||
LVEF | 0.08 | −0.08 | − 0.23 | −0.32§ | − 0.52§ | −0.49§ | − 0.20 | |||||
Thigh circumference | −0.41# | −0.14 | 0.13 | −0.17 | − 0.22 | −0.40# | − 0.02 | 0.32# | ||||
Rectus femoris thickness | −0.35# | −0.31* | 0.07 | −0.27# | − 0.41§ | −0.42§ | − 0.05 | 0.31# | 0.49§ | |||
Knee extensor strength | −0.36# | −0.21 | 0.05 | −0.36# | − 0.41§ | −0.60§ | − 0.14 | 0.43§ | 0.50§ | 0.69§ | ||
3-MH/Cr | 0.09 | 0.17 | 0.26* | 0.21# | 0.28# | 0.26# | 0.14 | −0.12 | −0.26* | −0.24* | − 0.22* | |
6-min walk distance | −0.19 | −0.14 | 0.10 | −0.42§ | − 0.77§ | −0.42§ | − 0.16 | 0.46§ | 0.23* | 0.32* | 0.37# | −0.28* |