The molecular co-chaperone Bcl2-associated athanogene 3 (Bag3) is a critical component of protein quality control (PQC) systems in the heart. Containing multiple protein binding
domains, Bag3 interacts in complex with numerous chaperones and clients to regulate protein folding, degradation, and response to intracellular protein aggregation. Mutations in Bag3 are associated with human cardiac myopathies, including a proline to leucine substitution at position 209 (P209L), which causes a particularly severe childhood-onset dilated cardiomyopathy. However, no therapies currently exist for Bag3-related cardiomyopathies and the molecular mechanisms by which the Bag3P209L mutation results in cardiomyopathy remain elusive. Here,
utilizing cardiac-specific Bag3P209L transgenic (Tg+) mice we investigated the signaling
mechanisms that result in Bag3-related cardiomyopathy. We discovered aberrant cardiac activation of proteotoxic p38 MAPK signaling, and demonstrated the efficacy of p38 inhibitors to both reduce cardiac proteotoxicity and restore cardiac function in Bag3P209L Tg+ mice. Our
results provide insight into the pathophysiology of Bag3P209L related cardiomyopathy and
identify p38 MAPK inhibition as a promising therapeutic target. Introduction
Bcl2-associated athanogene 3 (Bag3) is a 575 amino acid scaffolding protein consisting of multiple protein-binding domains including an n-terminal WW domain (21-55), two IVP motifs (87-101, 200-213), a proline-rich PxxP domain (302-412) and a c-terminal Bag domain
(420-499)102,103. Out of the six members in the Bag family, Bag3 is the only stress inducible
family member. Bag3 is evolutionarily conserved and plays important roles in maintaining proteostasis during stress-related cellular events and aging53. In fact, Bag3 has emerged as a
major regulatory hub to regulate multiple protein quality control pathways and mitigate intracellular protein aggregation55,59,67. Via its Bag domain, Bag3 canonically serves as a
nucleotide exchange factor for Hsc70/Hsp70 to facilitate protein refolding of misfolded protein clients104,105. Through binding with small heat shock proteins, including HspB8 and HspB6,
Bag3 plays a role in regulating chaperone-assisted selective autophagy58,106. Scaffolded Bag3
complexes coordinate aggresome formation62, chaperonin-dependent protein folding57, and stress
granule disassembly70. Additionally, Bag3 interacts with and mediates proteotoxic stress-induced
mitogen-activated protein kinase (MAPK) signaling67.
To date, over 30 distinct mutations in Bag3 are associated with disease characterized by cardiac and skeletal muscle myopathy, including a proline to leucine substitution mutation at position 209 (P209L) that causes a particularly severe childhood cardiac myopathy86–
88,90,91,94,95,107–109. The molecular mechanisms resulting in progressive Bag3-related myopathies
are largely unknown, however, the important role of Bag3 in protein quality control has led researchers to hypothesize that Bag3 mutations cause either a loss or alteration of Bag3 function, resulting in protein aggregation and proteotoxicity92,99,110,111. Consistent with this hypothesis,
expression of human Bag3P209L in cells results in the formation of cytoplasmic puncta, increased
Bag3P209L oligomerization, and an increase in stalled, insoluble cytosolic Bag3 client proteins,
indicative of small aggregate formation16,100. Small proteotoxic aggregates are increasingly
proteotoxicity has emerged as a disease driver in the heart, disease-specific proteotoxic signaling mechanisms that link proteotoxic aggregates to subsequent cardiac dysfunction remain largely unknown.
Our previous characterized cardiac-specific alphaMHC-Bag3P209L transgenic (Bag3P209L
Tg+) mice, a preclinical model that parallels the progressive cardiac dysfunction observed in human patients with autosomal dominant Bag3P209L mutations101. We identified toxic pre-
amyloid oligomers within cardiomyocytes in Bag3P209L Tg+ mice, presenting a unique
opportunity to both interrogate in vivo proteotoxic signaling and to test potential therapeutics for Bag3-related cardiomyopathies.
In the present study, we investigated aberrant signaling networks in Bag3P209L Tg+ mice
and identified p38/MAPK signaling as a major driver of observed proteotoxicity in Bag3P209L
Tg+ mice. The p38 signaling cascade is hyperactivated in numerous proteotoxic diseases
including Alzheimer’s disease, and p38 pathway inhibitors have garnered significant attention as potential therapeutics to treat proteinopathies73. In fact, p38 inhibitors tested in both pre-clinical
and clinical trials related to Alzheimer’s disease therapies, reduced proteotoxic aggregation and cellular dysfunction, leading to improved patient prognosis74,75. Here, we show that p38
inhibition in Bag3P209L Tg+ mice reversed cardiac dysfunction by 10 days of treatment and
reduced toxic pre-amyloid oligomers (PAOs) and intracellular aggregation of Bag3 client proteins. Together, these results point to the p38 pathway as a promising target for Bag3-related myopathies and potentially other cardiac myopathies characterized by intracellular
Results
The p38/MAPK pathway is activated in Bag3P209L Tg+ hearts
Very little is known about the underlying signaling networks that link mutations in Bag3 to the morphological features observed clinically in patients with myofibrillar myopathy and cardiomyopathy. Therefore, to uncover the signaling networks that contribute to the cardiac phenotype observed in Bag3P209L Tg+ mice, we utilized multiplexed kinase inhibitor bead
coupled Mass Spectrometry (MIB/MS), to investigate global activity changes in the kinome of Bag3P209L Tg+ hearts (Figure 2-1A). Following confirmation of systolic dysfunction, we
collected and lysed left ventricular cardiac sections of 12-month old Bag3P209L and Bag3 WT
mice (n=3 per group) and ran lysates in parallel over a column of immobilized kinase inhibitors with broad affinity towards activated kinases. Following elution and MS/MS we identified a total of 212 kinases in Bag3P209L Tg+ and Bag3 WT hearts, of which 12 kinases were differentially
activated in Bag3P209L Tg+ mice (Figure 2-1B) (Supplementary Figure 2-1A). Among the
differentially activated kinases were four metabolic kinases, which is not surprising given the metabolic changes known to occur under pathologically-induced cardiac dysfunction.
Interestingly, several key upstream signaling kinases in the p38/MAPK stress signaling cascade (Map2K4 and MAP3K5) were significantly increased in Bag3P209L Tg+ mice (>1.5 fold). Indeed,
Gene Ontology Enrichment Analysis of these kinases using the Panther pathway analysis tool, identified the p38/MAPK signaling pathway as one of the highest enriched pathways in
Bag3P209L Tg+ hearts (Table 2-1). By MIB/MS, only a moderate increase in p38/MAPK itself
was identified (Supplementary Figure 2-1B), however by western blotting we confirmed a 2.0- fold activation of p38 in Bag3P209L Tg+ compared to Bag3 WT hearts (Figure 2-1, C and D).
inactive p38 to the immobilized kinase inhibitor probes or overall kinase expression (Supplementary Figure 2-1). The p38/MAPK pathway is widely considered as a major proteotoxic signaling node and a potential therapeutic target in proteinopathies including Alzheimer’s disease72,113. We hypothesized that the p38/MAPK pathway is responsible for the
proteotoxicity and cardiomyopathy observed in Bag3P209L Tg+ mice.
p38/MAPK inhibition reverses systolic cardiac dysfunction in Bag3P209L Tg+ mice
To test our hypothesis that p38 signaling is an important regulator of proteotoxicity and cardiomyopathy observed in Bag3P209L Tg+ mice, we treated 12-month old Bag3P209L Tg+ mice
and Bag3 WT control mice with the p38 inhibitor SB203580 and measured cardiac function as our primary outcome. We investigated 4 groups of mice: 1) Bag3 WT mice receiving saline vehicle (n=3) 2) Bag3P209L Tg+ mice receiving saline vehicle (n=3) 3) Bag3 WT mice receiving
SB203580 (n=3) and 4) Bag3P209L receiving SB203580 (n=4). A dose of 1mg/ kg body of
SB203580 or saline vehicle control was administered daily for 30 days via intraperitoneal injection, in accordance with previous literature on SB203580 use in mice114. Baseline
echocardiography was performed on each of the groups prior to the study to confirm cardiac dysfunction in Bag3P209L Tg+ mice followed by echocardiography at 10, 20, and 30 days of
treatment. Remarkably, treatment with SB203580 not only prevented further functional impairment, but restored normal ejection fraction (EF) by 10 days which was sustained
throughout the course of treatment (Figure 2-2A). SB203580 did not alter body weight or heart rate in treated mice for the duration of the study (Table 2-2).
Although SB20380 is an adequate and widely used pharmacological p38 inhibitor in pre- clinical models, it is only a tool compound with limited clinical practicality due to its mediocre
inhibitor study on cardiac function, we sought to validate our findings using clinically-relevant, latest-generation p38 inhibitors that display improved pharmacokinetic profiles, improved specificity, and current usage in clinical trials116,117 (Table 2-3). Utilizing the p38 inhibitor Vx-
745, we performed a double-blind placebo controlled 30-day cross-over study and drug washout using eight Bag3P209L Tg+ and Bag3 WT mice. A dose of 10mg/ kg of Vx-745 was administered
b.i.d. via oral gavage for 30 days as preciously described76. Again, by 10 days, p38 inhibition
restored EF% by 10 days. Following 30-day treatment, drug washout resulted in a return of cardiac dysfunction (Figure 2-2B).
Utilizing the p38 inhibitor BCT-197, which boasts extremely low nanomolar affinity for p38, the same four treatment groups from the initial SB203580 study were used with an n=7 mice per group. A dose of 5 mg/ kg of BCT-197 delivered daily via oral gavage for 10 days was used as previously described in the literature118. BCT-197 treatment completely restored EF in
inhibitor-treated mice compared to Bag3 WT control mice.
Inhibitors of p38/MAPK reduce activated cardiac p38 signaling
The p38 inhibitors used in this study are all ATP-competitive inhibitors of active p38, resulting in the inability of phosphorylated p38 to activate direct downstream signaling targets, including MAPKAPK2. To test the signaling effects of 30-day treatment with SB203580 on
Bag3P209L Tg+ mice, we utilized MIB/MS to elucidate SB203580 mediated changes in the
activated kinome of treated Bag3P209L Tg+ and Bag3 WT mice compared to vehicle controls. We
identified 14 significant, differentially activated kinases including MAPKAPK2, as the most downregulated kinase upon inhibition (Figure 2-3A). Additionally, we detected a significant decrease in MAP3K5, and a moderate decrease in p38 itself (Supplementary Figure 2-1C)
consistent with a negative feedback loop in the p38 pathway after sustained, 30-day p38 signaling inhibition.
SB203580 also has low micromolar affinity for additional off-target kinases, which raises a potential argument that the reversal in cardiac dysfunction may be the result of off-target kinase inhibition, independent of p38 signaling119. However, there is no overlap of significantly
downregulated kinases in SB203580 treated Bag3P209L Tg+ mice with the HMS LINCs
KINOMEscan SB203580 kinase binding affinity database (ID:20177), indicating that the beneficial effects are likely dependent on p38 inhibition. This finding is also consistent with the functional studies performed using Vx-745 and BCT-197 which have minimal off-target kinase affinity and result in the restoration of cardiac function. Since BCT-197 is the most clinically advanced compound of the inhibitors tested, BCT-197 treated mice were used throughout the remaining biochemical experiments in this study. In fact, western blotting for activated phospho- p38 and downstream phospho-MAPKAPK2 identified almost complete p38 and MAPKAPK2 inhibition in BCT-197 treated Bag3P209L Tg+ (p<0.05) (Figure 2-3, B-D). Immunoblotting for
parallel MAPK pathways, SAPK/JNK and Erk1/2, confirm that inhibition was specific for the p38/MAPK signaling pathway (Supplemental Figure 2-1, D-F)
p38/MAPKinhibition reduces proteotoxic pre-amyloid oligomers
Inhibition of p38, preclinically in mouse and rat models of Alzheimer’s disease, and more recently in clinical trials for Alzheimer’s disease have been shown to reduce protein aggregation and proteotoxicity and restore neuronal function74–76. We previously hypothesized that cardiac
PAOs drive the disease pathology and dysfunction observed in Bag3P209L Tg+ mice. Therefore,
we wanted to determine if inhibiting p38 using BCT-197 decreased cardiac PAO levels. To measure the effect of p38 inhibition on proteotoxic pre-amyloid oligomers, we utilized a recently
developed in-gel staining technique, based on the widely used histological protein aggregation stain Thioflavin T, which recognizes amyloidogenic oligomers and short protofibrils120. First,
this approach identified an increase in amyloidogenic staining in heart lysates isolated from
Bag3P209L Tg+ mice compared to Bag3 WT mice. Next, we observed that BCT-197 treatment
was effective in lowering PAO levels in treated Bag3P209L Tg+ hearts, suggesting that p38
inhibition is sufficient to reduce PAOs in the heart (Figure 2-4 A, C). Utilizing an orthogonal technique, immunoblotting with a polyclonal A11 antibody (which recognizes pre-amyloid oligomers by a toxic epitope generated by a wide variety of different amyloidogenic
sequences)49, we again confirmed the reduction of PAOs in heart lysates isolated from Bag3P209L
Tg+ mice treated with BCT-197 (Figure 2-4 B, D). Interestingly, BCT-197 reduced PAOs in heart lysates from both treated Bag3P209L Tg+ and Bag3 WT control mice to levels lower than
vehicle treated Bag3 WT control mice. Previous studies demonstrated that p38 inhibition is protective in aging, even in the absence of functional decline and disease121,122. Since 16-month
old Bag3P209L Tg+ and Bag3 WT mice were used in this study, the effects we observed are
consistent with a cardioprotective role for p38 inhibition during aging in addition to a cardioprotective role during Bag3P209L associated cardiomyopathy.
Bag3P209L expands the PQC interaction network of wildtype Bag3 to include aggregate prone
proteins
In cells, a recent study has shown that mutant Bag3P209L traps Bag3-interacting PQC
proteins within non-functional, stalled, destabilized complexes which can manifest as PAOs or small insoluble aggregates leading to increased proteotoxicity and PQC collapse100. To test this
mechanism in vivo in Bag3P209L Tg+ mice, we first sought to elucidate changes in the binding
we performed in-gel affinity-purification coupled to MS on human FLAG-Bag3 WT and FLAG-
Bag3P209L transfected cos7 cells following immunoprecipitation for FLAG (Figure 2-5A)
(Supplementary Figure 2-2, A and B). We filtered the results by removing proteins with < 2 identified peptides and proteins that were < 2-fold of FLAG-GFP. Using that criteria, we identified a total of 43 Bag3 WT interactors including many known interacting partners (Figure B, C) Gene Set Enrichment analysis on Bag3 interacting proteins, using the Panther pathway analysis tool, identified proteins that were largely involved in PQC processes and cytoskeletal organization. Remarkably, we identified 261 interacting proteins of Bag3P209L including almost
complete overlap (37 of 43) with Bag3 WT including HSP70 subunits and HSPB8 (Figure 2-3, B and D). The overlapping proteins were largely unsurprising as the P209L mutation is known to have no detrimental effect on binding to Hsp70 or HspB8. However, a subset of shared
interactors including filamin-c (FLNC), and tubulin alpha and beta subunits (TUBA1C and TUBB) had a > 2-fold increase in binding to Bag3P209L which was confirmed in an independent
MS experiment (Supplemental Figure 2-2, C and D). FLNC and Tubulin are major components of cardiac aggregates in myofibrillar and cardiac myopathies.
Surprisingly, the largest group of proteins identified were 224 proteins that interacted with Bag3P209L but not Bag3 WT. Included in this protein set were numerous small heat shock
proteins involved in aggregate clearance (CRYAB, DNAJA1, DNAJA2), known markers of aggregation and stress granules in proteinopathies (TDP-43), proteasome subunits, and ribonucleoprotein complex interactors (Figure 2-5D). Select proteins of interest from Affinity- MS were confirmed using coimmunoprecipitation and subsequent immunoblotting, including shared PQC interactors (HspB1/Hsp27 and HspB8) and Bag3P209L alone interactors (TDP-43 and
itself as an interacting partner to wildtype Bag367, and we identified several p38 interacting
partners that bind to both Bag3 and Bag3P209L (Figure 2-3 D, E). Since p38 stress signaling is
important in regulating proteotoxic signaling, we hypothesized that p38 itself may have an increased interaction with Bag3P209L. Indeed, while both Bag3 and Bag3P209L interact with HA-
p38 in co-transfected cos7 cells, Bag3P209L interacts more strongly with p38 (Figure 2-5G)
(Supplementary Figure 2-2E). Further investigation into the consequences of this direct interaction are warranted, however Bag3 and Bag3P209L are phosphorylated in the presence of
p38 suggesting Bag3 may be a p38 phosphorylation target (Supplementary Figure 2-2F). Intriguingly, not all PQC and proteotoxic signaling proteins interact more strongly with Bag3P209L, such as Gadd34 and PP1, two known Bag3 interacting proteins that regulate the
unfolded protein response56 (Supplementary Figure 2-2E). Decreased levels of GADD34 and
PP1 are co-immunoprecipitated with Bag3P209L compared to Bag3, indicating that the P209L
mutation may contribute some loss-of-function as well as its proposed toxic gain-of-function protein trapping.
p38 MAPK inhibition reduces insoluble cardiac Bag3 PQC partners and uncleared ubiquitinated proteins
To test whether Bag3P209L client proteins were changed in Bag3P209L Tg+ hearts, we
immunoblotted for Bag3-interacting molecular clients. In whole ventricle lysates the steady-state levels of Bag3P209L interacting partners were largely unchanged (Figure 2-6, A and B). Notably,
only levels of CryAB were lower in Bag3P209L Tg+ hearts. Next, since we hypothesized that
Bag3P209L traps interacting partners within small insoluble aggregates, we isolated the triton-x
insoluble, myofilament enriched fraction from Bag3P209L Tg+ and Bag3 WT hearts. Strikingly, 5
(Figure 2-6, C and D). The proteins with the highest increase in the insoluble fraction (TDP-43, CryAB, and DNAJA1) were proteins that interact preferentially with Bag3P209L over wildtype
Bag3.
Next, we tested whether inhibition of cardiac p38 signaling is able to reduce the presence of insoluble Bag3 interacting partners found within the insoluble fraction of Bag3P209L Tg+
hearts. Indeed, p38 inhibition reduced every Bag3P209L client protein tested within the insoluble
fraction (Figure 2-6, C and D). DNAJA1, HspB8, HspB6 and Hsp27 were normalized to levels found in vehicle-treated Bag3 WT hearts, while TDP-43 and CryAB were reduced, but not completely to Bag3 WT levels.
Since Bag3P209L clears ubiquitinated clients via chaperone-mediated autophagy, one
marker of Bag3P209L induced PQC collapse is an accumulation of insoluble ubiquitinated
proteins. To test whether insoluble ubiquitinated proteins were increased in Bag3P209L Tg+
hearts, we isolated total proteins and the myofilament enriched insoluble fraction from Bag3P209L
Tg+ and Bag3 WT hearts and blotted for ubiquitin. We found an increase in total ubiquitinated proteins in both total protein lysates (Figure 2-7, A and B) and the myofilament enriched fraction (Figure 2-7, C and D), indicative of PQC impairment and collapse. Cardiac p38 inhibition
successfully reduced ubiquitinated proteins to Bag3 wildtype levels. Discussion
Although mutations in Bag3 are known to result in childhood cardiac and skeletal muscle myopathies, little is known about the cellular signaling mechanisms whereby Bag3 mutations result in PQC disruption and cardiomyopathy. Several groups have identified a Bag3P209L
mutation which causes a particularly severe, childhood-onset cardiomyopathy and
we aimed to elucidate targetable signaling pathways underlying disease pathology. Specifically, we identified the p38/MAPK signaling pathway as a key cardiac signaling pathway in Bag3P209L
Tg+ hearts, linking observed cardiac proteotoxicity and dysfunction. We then demonstrated the potential of p38 pathway inhibitors to treat the disease.