Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Iron-regulatory proteins secure iron availability in skeletal muscle to preserve muscle mass and exercise tolerance
B. M. Chung1, W. Jonas2, F. Rostami1, Z. Malik1, M. Szaroszyk1, J. T. Thackeray3, M. Ost2, J. Hegermann4, F. Bengel3, J. Bauersachs5, J. Heineke6, K. C. Wollert1, A. Schürmann2, T. Kempf5
1Molekulare und Translationale Kardiologie, Medizinische Hochschule Hannover, Hannover; 2Experimentelle Diabetologie, Deutsches Institut für Ernährungsforschung Potsdam-Rehbrücke (DIfE), Nuthetal; 3Klinik für Nuklearmedizin, Medizinische Hochschule Hannover, Hannover; 4Zentrale Forschungseinrichtung Elektronenmikroskopie, Medizinische Hochschule Hannover, Hannover; 5Kardiologie und Angiologie, Medizinische Hochschule Hannover, Hannover; 6Kardiovaskuläre Physiologie, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim;
Iron deficiency (ID) is a frequent comorbidity in heart failure (HF) associated with exercise intolerance and increased mortality. In HF, tissue iron content is unrelated to systemic iron status, indicating that tissue iron homeostasis, e.g. in heart and skeletal muscle, is independently regulated. We here show that HF impairs iron regulatory protein (IRP) 1 and 2 activity in skeletal muscle, thereby promoting skeletal muscle ID. To specifically explore the functional implications of skeletal muscle IRP inactivation and ID, we generated mice with skeletal muscle-restricted deletion of IRP1 and 2.
 
First, we analysed skeletal muscle iron homeostasis in C57BL/6J male mice with transverse aortic constriction (TAC)-induced HF. Ninety days after TAC, IRP-iron responsive element (IRE) binding activity was reduced in M. gastrocnemius (73±4% vs. sham, P<0.01), resulting in reduced transferrin receptor 1 expression (Tfr1, required for iron uptake, 55±13%, P<0.05), reduced non-heme iron content (62±4%, P<0.01), and diminished mitochondrial complex I abundance (required for oxidative phosphorylation, OXPHOS) (50±4%, P<0.01).
 
Next, we generated skeletal muscle-selective Irp1/2 targeted mice (Cre-Irp1/2f/f) by crossing Irp1/2 floxed mice (Irp1/2f/f) with mice expressing Cre recombinase in skeletal muscle under the control of the myosin light-chain 1 promoter (MLC1-Cre). IRP-IRE binding activity in M. gastrocnemius was significantly decreased Cre-Irp1/2f/f mice (52±5% vs. Irp1/2f/f, P<0.01). Moreover, Tfr1 expression (19±5%, P<0.01) and non-heme iron concentration (59±4%, P<0.001) were reduced in skeletal muscle from Cre-Irp1/2f/f mice, while systemic iron status and iron content in other organs were well preserved. Body weight from both genotypes was not different until 13 weeks of age. Muscle masses and fiber diameters were smaller in skeletal muscle of Cre-Irp1/2f/f compared to Irp1/2f/f mice. Complex I and II expression was massively decreased in skeletal muscle from Cre-Irp1/2f/f mice (6±5% and 6±2%, respectively, P<0.01 vs. Irp1/2f/f).  High-resolution respirometry of muscle fibers obtained from M. soleus revealed a lower ex vivo mitochondrial respiratory capacity in Cre-Irp1/2f/f mice, indicating inefficient mitochondrial oxidative function and energy production. On the other hand, skeletal muscle glycolysis was enhanced in Cre-Irp1/2f/f mice as indicated by greater uptake of 18F-desoxyglucose (M. quadriceps: 150±16%, P<0.05, vs. Irp1/2f/f) and faster plasma glucose clearance. Exercise performance was strongly decreased in Cre-Irp1/2f/f mice (9±1% in distance and 40±2% in maximum speed vs. Irp1/2f/f, P<0.001). Transcriptome analyses revealed an upregulation of endoplasmic reticulum stress-, atrophy-, mitochondrial dysfunction-, and inflammation-associated gene clusters in M. quadriceps from Cre-Irp1/2f/f mice. Electron microscopy showed swollen mitochondria and sarcoplasmic reticulum in M. soleus and M. extensor digitorum longus of Cre-Irp1/2f/f mice. Intraperitoneal iron supplementation normalized skeletal muscle iron content and restored oxidative phosphorylation in Cre-Irp1/2f/f mice.
 
In conclusion, skeletal muscle IRP-IRE binding activity is reduced in HF, resulting in skeletal muscle ID. As shown in Cre-Irp1/2f/f mice, skeletal muscle-selective ID results in mitochondrial dysfunction, muscle atrophy, and exercise intolerance. 
 
 

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