Cardiomyocyte Contractile Impairment in Heart Failure Results from Reduced BAG3-mediated Sarcomeric Protein Turnover

Abstract

Compromised force-generating capacity is a hallmark of heart failure both at the 1 organ 1 and single-cell level 2-6 . This is primarily due to changes at the sarcomere 7 , the 2 functional unit in cardiomyocytes responsible for contraction. However, the 3 mechanisms of sarcomeric force depression in heart failure are incompletely 4 understood. We show in human heart failure that myofilament BAG3 levels predict the 5 severity of sarcomere dysfunction and in a mouse heart failure model, increasing BAG3 6 expression rescues contractile function. Further, myofilament ubiquitin increases in 7 heart failure, indicating impaired protein turnover, but is reduced with increased BAG3 8 expression. Mass spectrometry revealed Hsp70, HspB8 associate with myofilament 9 BAG3, forming a conserved selective autophagy complex, which localizes to the Z-disc. 10Assembly of this complex at the sarcomere is BAG3-dependent and triggered by 11 proteotoxic stress, however, its clearance stalls in heart failure. Together, these 12 findings identify BAG3-dependent autophagy as essential for functional maintenance of 13 the cardiac sarcomere. 14 Word Count: 150/150 15 Cardiomyocytes isolated from the left ventricle (LV) of dilated cardiomyopathy (DCM) 16 heart failure patients (Extended Data Table 1) displayed a reduction in myofilament maximum 17 calcium-activated force (Fmax) and increased calcium sensitivity (decreased EC50 -calcium 18 concentration required to elicit half maximal force) compared with non-failing donors (Fig. 1a-19 c). Changes in site-specific phosphorylation of myofilament proteins, most commonly troponin 20 I, are widely attributed to this expected increase in calcium sensitivity found in heart failure 8 .21The mechanisms underlying the decreased Fmax, which describes the inherent force-22 generating capacity of a cardiomyocyte, are less well defined. One possible explanation for 23 decreased Fmax in heart failure is that there are fewer functional sarcomeres and thus, 24 irrespective of calcium concentration, the ability to produce force is impaired 9 . However, the 100 of sarcomere proteins through CASA. 101To test whether the assembly of this complex in the myofilament fraction was BAG3-102 dependent, we used mouse models of cardiomyocyte-specific heterozygous and homozygous 103 BAG3 deletion 24 . Western blot of myofilament-enriched LV tissue revealed myofilament levels 104 of HspB8, but not Hsp70, were significantly reduced in the partial absence of BAG3 (BAG3 +/-, 105 20% reduction of myofilament BAG3 vs. wild-type) and barely detectable in the BAG3 -/-106 hearts (Fig. 3a). The same relationship was found in human DCM samples, where samples 107 with lowest myofilament BAG3 (≥20% decreased relative to non-failing) had significantly 108 reduced HspB8 levels but not Hsp70 levels (Fig. 3b-c). Notably, the E3 ubiquitin ligase for 109 Hsp70 clients, CHIP, was also significantly reduced with decreased BAG3 expression 110 (Extended Data Fig. 2). These data indicate that BAG3 is required for full assembly of the 111 CASA...

Transthyretin deposition alters cardiomyocyte sarcomeric architecture, calcium transients, and contractile force

“…During the loss of sarcomere content seen with TTR, one must expect filament disassembly generating an excess of unassembled proteins, which are known in other situations to become tagged for degradation and reprocessing (Khalil & Xiao, 2018). For example, myocardial tissue from patients with dilated cardiomyopathy has increased levels of myofibrillar ubiquitination associated with dysfunctional protein degradation through autophagy via the co‐chaperone BAG3 (Martin & Kirk, 2020; Martin et al, 2021). Loading stabilizes the sarcomeric structure and decreases the rate at which proteins are exchanged with the cytoplasm (Russell & Solís, 2021; Solís & Russell, 2019).…”

Functional analysis of a common BAG3 allele associated with protection from heart failure

“…A similar approach has been used to study small molecule perturbations on iPS-CMs and other cell types 41,42 . The use of iPS-CMs as a model for cardiomyopathies is not without limitations: iPS-CMs are known to lack some important features of mature myocytes and are not exposed to mechanical stress from pressure load, which is a predominant factor in the challenging of the myocardial protein quality control machinery 28,[43][44][45] . This last point could influence the profile of BAG3 interactions and might explain the lower representation of structural and sarcomeric proteins in our BAG3 interactome compared to other similar studies using primary heart tissue 27,28 .…”

“…In cardiac and skeletal muscle, a functioning protein quality control network is essential to dynamically maintain myofibrillar structures through constant cycles of contraction 24 . Accordingly, BAG3 impairment results in heart and skeletal muscle pathology [25][26][27][28] . These studies indicate the BAG3 protein has an essential role in muscle, particularly in cardiomyocytes, which lack replicative potential.…”

GSK-3β Localizes to the Cardiac Z-Disc to Maintain Length Dependent Activation

“…P <0.05 were considered statistically significant. A priori power analyses were performed based on data from published studies 13,37,38 and pilot experiments.…”