Establishment of a model of heart failure and sarcopenia
Anterior descending artery ligation combined with hindlimb unloading was used to establish the model of HF with sarcopenia. Compared with the control and sham group, the HF group showed significantly decreased EF and FS (P < 0.0001 for both, Fig. 1A) and significantly increased serum BNP level (P < 0.05, Fig. 1B), indicating that we have successfully constructed a HF model.
Serum CK level in the HF group was higher (P < 0.0001, Fig. 1B) than that in the control and sham group. The HF group had significantly reduced forelimb grip strength, hanging impulse, maximum running time, and maximum running distance, indicating that exercise capacity of the HF group declined (P < 0.0001, Fig. 1C). To detect changes in skeletal muscle content, we calculated the ratio of gastrocnemius muscle weight to tibial length and discovered that this ratio in the HF group was significantly lower than that in the control and sham group (P < 0.05, Fig. 1C). HE staining showed that skeletal muscle fibers' arrangement in the HF group was disordered and that the CSA was significantly reduced (P < 0.0001, Fig. 1C). In the HF group, skeletal muscles were damaged, muscle fibers were atrophic, and skeletal muscle function was weakened. Hindlimb unloading based on HF further aggravated skeletal muscle damage. Compared with the HF group, the HF + sarcopenia group has higher CK level (P < 0.0001, Fig. 1B), lower exercise capacity (P < 0.01, Fig. 1C), and smaller skeletal muscle CSA (P < 0.01, Fig. 1C).
Besides, compared with mice in the HF group, both the BNP level and the LDH level of mice in the HF + sarcopenia group were increased significantly (P < 0.05 and P < 0.01, respectively, Fig. 1B). It shows that the hindlimb unloading further aggravates skeletal muscle atrophy and leads to an aggravation of HF. In summary, we successfully established a mouse model of HF with sarcopenia through ligation of the anterior descending branch combined with hindlimb unloading.
Levosimendan improved cardiac function and exercise capacity in mice with heart failure and sarcopenia
Mice were injected intraperitoneally of 3mg/Kg levosimendan. The HF + levosimendan group had higher EF and FS compared with the HF group. After injection of levosimendan, EF (P < 0.0001, Fig. 2B) and FS (P < 0.01, Fig. 2C) of the HF + sarcopenia group increased, the cardiac function was significantly improved. It shows that 3mg/Kg levosimendan was sufficient to enhance the cardiac function of mice with HF and sarcopenia.
The exercise test showed that compared with the HF group, the HF + levosimendan group had significantly increased forelimb grip strength (P < 0.001, Fig. 2D), hanging impulse (P < 0.0001, Fig. 2E), maximum running time (P < 0.01, Fig. 2F), maximum running distance (P < 0.01, Fig. 2G), and ratio of gastrocnemius muscle weight to tibial length (P < 0.0001, Fig. 2H) indicating improved exercise capacity and skeletal muscle content of the mice in the HF + sarcopenia group after intervention with levosimendan (P < 0.0001, Fig. 2C-G). Levosimendan has an antagonistic effect on sarcopenia (P < 0.05). After correcting for EF, levosimendan could still improve the grip strength (P < 0.0001), hanging impulse (P < 0.05), and maximum running distance (P < 0.0001) of mice with HF and sarcopenia, suggesting that the improvement of levosimendan on exercise capacity is achieved independently of improved cardiac function. The above results indicate that levosimendan could increase myocardial contractility and cardiac function in mice with HF and sarcopenia, as well as skeletal muscle function.
levosimendan improved myofiber atrophy in mice with heart failure and sarcopenia
HE staining showed that the HF + levosimendan group had a more regular myofiber structure (Fig. 3A) and increased CSA (P < 0.01, Fig. 3B) compared with the HF group. Muscle fiber atrophy in the HF + sarcopenia group was improved after levosimendan intervention (P < 0.0001, Fig. 3B), indicating that levosimendan enhanced exercise capacity of mice with heart failure and sarcopenia by improving skeletal muscle atrophy. After correcting for EF, levosimendan could still increase the CSA (P < 0.0001), suggesting that levosimendan's effect to inhibit the decrease of CSA caused by heart failure and sarcopenia was achieved independently of improved heart function.
levosimendan promoted slow muscle fiber differentiation in mice with heart failure and sarcopenia
Furthermore, muscle fiber typing (Fig. 3A) was evaluated. Compared with the control group, the HF group had a significantly increased proportion of fast muscle fibers (P < 0.0001, Fig. 3C), a decreased proportion of slow muscle fibers (P < 0.01, Fig. 3D), and drastically reduced ratio of slow to fast muscle fibers (P < 0.05, Fig. 3E). Based on HF, hindlimb unloading further affected the ratio of muscle fibers. Compared with the HF group, the HF + sarcopenia group exhibited increased content of fast muscle fibers, decreased content of slow muscle fibers, and decreased ratio of slow to fast muscle fibers (P < 0.0001, Fig. 3C-E). Compared with the HF group, the ratio of fast muscle fibers was decreased, the ratio of slow muscle fibers was increased, and the ratio of slow to fast muscle fibers was raised in the HF + levosimendan group (P < 0.0001, Fig. 3C-E). The ratio of slow to fast muscle fibers in the HF + sarcopenia group was also improved after injection of levosimendan (P < 0.0001, Fig. 3C-E), indicating that levosimendan could also enhance exercise capacity of mice with heart failure and sarcopenia by influencing muscle fiber typing. After correcting for EF, levosimendan's effect on muscle fiber typing disappeared (P > 0.05), suggesting that the improvement of muscle fiber typing might be attributed to improved cardiac function.
Levosimendan improved mitochondrial function of skeletal muscle
The most fundamental difference between skeletal muscle fast-twitch and slow-twitch fibers is the distribution of mitochondria, so mitochondrial function was examined next. Compared with the control group, the HF group had less mitochondrial content (P < 0.05, Fig. 4A) and reduced membrane potential (P < 0.0001, Fig. 4B), indicating that HF reduced the content of mitochondria and integrity of functions. Under conditions of HF, hindlimb unloading caused mitochondrial dysfunction. Mitochondrial content (P < 0.0001, Fig. 4A) and mitochondrial membrane potential (P < 0.0001, Fig. 4B) of the HF + sarcopenia group were lower than those of the HF group. Compared with the HF group, the HF + levosimendan group had a significant increase in mitochondrial content (P < 0.05, Fig. 4A) and mitochondrial membrane potential (P < 0.0001, Fig. 4B). Mitochondrial content (P < 0.0001, Fig. 4A) and mitochondrial membrane potential (P < 0.0001, Fig. 4B) of the HF + sarcopenia group were significantly increased after injection of levosimendan. Levosimendan had an antagonistic effect on sarcopenia (P < 0.05). After correcting for EF, levosimendan could still increase mitochondrial content (P < 0.0001) and reduce the loss of membrane potential, suggesting that levosimendan could improve skeletal muscle mitochondrial function independently of improvement of cardiac function. The above results indicated that levosimendan could protect mitochondrial function and improve skeletal muscle function.
Levosimendan improved skeletal muscle apoptosis
The destruction of mitochondrial membrane integrity will induce apoptosis, so we further tested the level of skeletal muscle apoptosis. Western blotting showed that compared with the control group, the HF group had increased level of BAX (P < 0.05, Fig. 5D) and decreased level of BCL2 (P < 0.0001, Fig. 5E). But cleaved caspase-9 (P > 0.05, Fig. 5B), cleaved caspase-3 (P > 0.05, Fig. 5A) had no significant changes, indicating that under HF conditions, the skeletal muscle did not undergo substantial apoptosis. Based on HF, hindlimb unloading increased the level of skeletal muscle apoptosis. Compared with the HF group, the expression levels of cleaved caspase-9, cleaved caspase-3, and BAX in the HF + sarcopenia group were significantly increased (P < 0.0001, Fig. 5C-D), but BCL2 did not change significantly (P > 0.05, Fig. 5E). After injection of levosimendan, cleaved caspase-9 (P < 0.0001, Fig. 5B), cleaved caspase-3 (P < 0.01, Fig. 5B), and BAX expression decreased significantly (P < 0.0001, Fig. 5D), the BCL2 expression level increased significantly (P < 0.05, Fig. 5E). TUNEL staining results (Fig. 5F-G) were consistent with Western blotting results, indicating that levosimendan could improve skeletal muscle function by reducing the apoptosis of skeletal muscle. After correcting for EF, levosimendan could still reduce cleaved caspase-9 (P < 0.0001) and BAX expression (P < 0.0001), increase BCL2 expression (P < 0.0001), and reduce apoptosis rate (P < 0.01). It is suggested that the improvement of skeletal muscle apoptosis by levosimendan was independent of improved cardiac function.
Possible causes of apoptosis
Both oxidative stress and chronic inflammation can induce and aggravate apoptosis level, so we next tested the level of oxidative stress and inflammation of skeletal muscle. The content of GSH, GSSG, and SOD in skeletal muscle were tested. Compared with the control group, GSH/GSSG (P < 0.01, Fig. 6A) and SOD content decreased in the HF group (P < 0.05, Fig. 6B), indicating that HF had aggravated the oxidative stress level of skeletal muscle. In the condition of HF, hindlimb unloading deteriorated the level of oxidative stress further. GSH/GSSG (P < 0.001, Fig. 6A) and SOD content of HF + sarcopenia group significantly were lower than those of the HF group (P < 0.005, Fig. 6B). Compared with the HF group, the GSH/GSSG (P < 0.0001, Fig. 6A) and SOD content of the HF + levosimendan group increased significantly (P < 0.05, Fig. 6B). GSH/GSSG (P < 0.0001, Fig. 6A) and SOD (P < 0.05, Fig. 6B) levels of the HF + sarcopenia group were significantly increased after injection of levosimendan, and the oxidative stress was also alleviated. Levosimendan had an antagonistic effect on sarcopenia (P < 0.05). After correcting for EF, this improvement effect of levosimendan disappeared (P > 0.05), suggesting that the improvement of skeletal muscle oxidative stress by levosimendan might be attributed to improved heart function.
Immunohistochemical staining (Fig. 6E) was next performed to investigate the inflammation level of skeletal muscle. Compared with the control group, the expression levels of IL-6 (P < 0.05, Fig. 6C) and TNF-α (P < 0.05, Fig. 6D) of skeletal muscle in HF group increased, indicating that the level of chronic inflammation of skeletal muscle was elevated under HF conditions. Based on HF, hindlimb unloading worsened the level of skeletal muscle inflammation. The expression of inflammatory factors in the HF + sarcopenia group was higher than those in the HF group (P < 0.0001, Fig. 6C-D). Compared with the HF group, the expression of IL-6 (P < 0.05, Fig. 6C) and TNF-α (P < 0.0001, Fig. 6D) in the HF + levosimendan group was significantly decreased, and the inflammation level of the HF + sarcopenia group was effectively improved after injection of levosimendan (P < 0.0001, Fig. 6C-D). Levosimendan had an antagonistic effect on sarcopenia (P < 0.05). After correction for EF, Levosimendan could still reduce the expression of IL-6 (P < 0.01) and TNF-α (P < 0.01), indicating that the improvement of skeletal muscle inflammation by levosimendan was independent of improved cardiac function. It’s suggested that levosimendan could improve the apoptosis level of skeletal muscle by reducing the inflammation level of skeletal muscle.