Regular ArticleDysregulated autophagy in restrictive cardiomyopathy due to Pro209Leu mutation in BAG3
Introduction
Myofibrillar myopathies (MFM) are a group of progressive hereditary neuromuscular disorders, which typically have a late onset and are frequently associated with cardiac involvement [1]. Pathogenic mutations in several different genes were found to be associated with MFM, including DES (desmin), CRYAB (αB-crystallin, also known as HSPB5, heat shock family B member 5), MYOT (myotilin), LDB3 (LIM domain binding 3, also known as ZASP, Z-band alternatively spliced PDZ containing protein), FLNC (filamin C), FHL1 (four and a half LIM domains 1), TTN (titin), and BAG3 (bcl-2 associated athanogene 3). These proteins are located at the Z-disks and pathogenic mutations affecting them can result in protein aggregation and disintegration of myofibrillar architecture [2], [3], [4], [5], [6], [7], [8], [9], [10]. In some instances, the protein aggregates show specific morphologic characteristics indicative of the underlying mutation [11].
Mutations in BAG3 are a rare cause of MFM. A proline to leucine mutation in the second IPV domain of BAG3 (BAG3-Pro209Leu) is associated with rapidly progressive restrictive cardiomyopathy (RCM), often requiring heart transplantation during childhood. Additional symptoms such as proximal myopathy, neuropathy, rigid spine and ventilatory insufficiency may precede or succeed the clinical signs of RCM [12], [13], [14], [15], [16], [17] (Table 1). In some patients with Pro209Leu mutation, light microscopic examination showed intracytoplasmatic inclusions in skeletal muscle containing the Z-disk proteins desmin and αB-crystallin, and electron microscopic examination revealed disintegration and disarray of myofibrils with aggregates of degraded filaments and electron dense material [13], [14], [16].
Autophagy in striated muscle is a complex process that involves different pathways and is crucial for protein homeostasis during tension-induced stress [22], [23], [24]. The autophagy cascade can be upregulated due to various forms of cellular stress and is dysregulated in different diseases [20], [25], [26]. BAG3 plays a key role in selective macroautophagy. Although expressed in various tissues, BAG3 is particularly abundant in mechanically strained skeletal and cardiac muscle cells [18]. It is necessary for the Z-disk integrity and is important for autophagy of damaged or misfolded protein in striated myocytes [19], [20], [21]. BAG3 is upregulated under mechanical stress and induces the formation of a multicomponent heat shock machinery at the Z-disk involving heat shock protein (Hsp70) and small heat shock proteins (HSPBs), necessary for delivery of targeted proteins to autophagosomes [27], [28], [29], [30], [31], [32], [33], [34], [35]. In addition, BAG3 regulates local mTORC1 function and is involved in filamin protein synthesis [22], [36]. In the heart, it protects cardiomyocytes against proteotoxic stress and mutation in the BAG3 gene has been associated with sarcomere disarray and reduced contraction power in cardiomyocytes, as well as with dilated cardiomyopathy [21], [37].
Taking into account the multiple functions of BAG3 in controlling protein homeostasis and autophagy in striated muscle, a mutation in the gene may result in a defect of myocyte function. In line with this notion, Bag3-deficient mice develop fulminant myofibrillar myopathy with early lethality [18], [38]. In zebrafish, overexpression of the mutant BAG3-Pro209Leu results in aggregate formation, while loss of wild type BAG3 causes myofibrillar disintegration [39].
In contrast to skeletal muscle, morphologic alterations in cardiac tissue have not been analyzed in detail [12], thereby limiting the pathophysiological understanding of the BAG3-Pro209Leu-related RCM and impeding the development of new therapeutic strategies. Here, we performed comprehensive morphological and biochemical analyses in explanted heart tissue from a patient with RCM caused by the BAG3-Pro209Leu mutation, focusing on the assessment of protein aggregation, myofibrillar structure and autophagy.
Section snippets
Cardiac muscle analysis
This work was approved by the Ethics Committee of the University of Giessen and a written informed consent was obtained from all parents. Tissue samples from the patient's explanted heart (P) and from two controls were analyzed. One control tissue was from an eight-year-old child with pulmonary atresia with intact ventricular septum (intraoperative biopsy) (C1), and one was an explanted heart from a one-year-old child with hypoplastic left heart syndrome (C2).
Next generation sequencing
To identify the causative gene,
Case history
The patient is the second child born to healthy non-consanguineous German parents. At age four years, a heart murmur was noticed. A subsequent echocardiogram was suggestive of restrictive cardiomyopathy, later confirmed by MRI (Fig. 1) and endomyocardial biopsy. The patient's cardiac function remained stable until age six years, when he started complaining about dyspnea during minor exertion. In addition, nocturnal hypoxemia and hypercapnia were detected, necessitating non-invasive assisted
Discussion
Myofibrillar myopathies (MFM) are a subgroup of progressive neuromuscular disorders characterized morphologically by typical intracytoplasmic inclusions, containing proteins associated with myofibril disintegration, and by Z-disk alterations [2], [4], [7], [9], [59]. Similar morphological features have been described in patients with MFM caused by the BAG3-Pro209Leu mutation. However, the clinical symptoms in the latter differ from those seen in other forms of MFM, as the BAG3-Pro209Leu-related
Conclusions
In conclusion, we present the first detailed morphological and biochemical characterization of cardiac tissue in a patient with severe MFM caused by the BAG3-Pro209Leu mutation. BAG3-mutant cardiac tissue showed protein aggregates and myofibrillar disintegration typical for MFM. The morphological alterations were similar, but more severe than those described in skeletal muscle samples, and are consistent with a severe cardiomyopathy. Our results support the hypotheses that dysregulation of
Acknowledgement
The authors thank the patients and their families for their cooperation and permission to publish the data. The authors thank Hannah Schlierbach, Jonas Görlach and Kerstin Leib for their excellent technical assistance.
Conflicts of interest
The authors declare that they have no conflict of interest.
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