Particularly in terminally-differentiated cells that are unable to readily regenerate, including neurons and cardiomyocytes, PQC processes are responsible for sustained regulation of the proteome throughout the entire lifetime of an organism. Under basal conditions and in response to physiologic environmental stresses, PQC machinery coordinate protein folding, protein degradation, and cellular response to misfolded protein aggregates without disrupting normal cellular functions1. However, under pathologic stresses, PQC processes can be disrupted,
resulting in proteotoxicity and progressive PQC collapse. This mechanism has long been considered the underlying molecular phenotype responsible for Alzheimer’s disease and numerous other proteinopathies in the brain186.
More recently, proteotoxicity is increasingly recognized to contribute to disease pathology in the heart. Specifically, the accumulation of misfolded proteins and subsequent cardiac proteotoxicity has been implicated as a driver of pathology in myofibrillar myopathies, a group of heterogenous disorders characterized by cardiac and skeletal muscle wasting112. Causal
mutations in several PQC proteins including Bag3, result in myofibrillar myopathy with varying degrees of skeletal and cardiac muscle dysfunction4. To date, over 30 mutations in Bag3 have
been identified and many, including a P209L mutation, result in particularly severe, childhood onset cardiomyopathies187. Utilizing a recently characterized, cardiac-specific Bag3P209L Tg+
therapeutic targets. First, in Chapter Two, I apply MIB/MS kinome profiling to illuminate p38 MAPK signaling as a regulator of Bag3P209L induced cardiac dysfunction and demonstrate the
utility of p38 inhibitors to treat Bag3-related cardiomyopathy. Then, in Chapter Three, I identify mitochondrial dysfunction, NAD handling, and cardiac protein hyperacetylation as secondary pathologies in Bag3P209L Tg+ mice. In this chapter, I consider how these observations can guide
future investigations.
Cardiac proteotoxic p38 signaling in Bag3P209L Tg+ mice
The molecular mechanisms underlying Bag3-related cardiac myopathies are largely unknown. Although numerous mutations have been identified in Bag3 leading to progressive cardiac dysfunction, very few mutations have been modeled preclinically142. The P209L
mutation in Bag3 has been reported in at least five families, and the effects of Bag3P209L in cells
and zebrafish have identified protein aggregation16,99. Recently, our laboratory generated and
characterized the first pre-clinical mouse model of Bag3-related cardiomyopathy, a cardiac- specific Bag3P209L Tg+ mouse101. In the present study, I identified PAOs and subsequent
proteotoxicity, resulting in small insoluble aggregates as a driver of cardiac dysfunction,
providing the first hard evidence linking PAOs to Bag3-related cardiomyopathy. Furthermore, I identified the p38 MAPK signaling cascade as a predominant regulator of PAO-induced cardiac proteotoxicity. These studies provide a mechanistic framework that can be used to determine whether other Bag3-related cardiomyopathies share the same pathobiology. Recently, a novel mouse model of Bag3E455K cardiomyopathy was generated and small insoluble protein
aggregates were identified, although PAOs were not directly measured80. Future studies,
investigating Bag3E455K mice for PAOs and aberrant p38 signaling, will determine whether
additional pre-clinical models of Bag3 myopathy-causing mutations are warranted to determine whether mutation-specific pathology is generalizable among all Bag3-related myopathies.
In addition to Bag3-related cardiomyopathy, cardiac and skeletal muscle myopathies caused by other PQC proteins may result in shared proteotoxic signaling. Although very few models of myofibrillar myopathies exist today, mouse models of both Desmin and CryAB- related myopathies exhibit cardiac PAOs46,47. This suggests that potential overlap exists in
proteotoxicity downstream of PAO formation. It is known that proteotoxic p38 signaling can be induced by PAOs in neurodegenerative proteinopathies caused by distinct genetic mutations72,126.
Therefore, measuring p38 signaling cascades in other cardiac myopathies where PAOs are
present may further demonstrate the applicability of my findings to other cardiac proteinopathies. Inhibition of p38 as a therapeutic for Bag3-related cardiomyopathy
In neurodegenerative proteinopathies, proteotoxic p38 signaling is a critical regulator of disease pathology, and p38 inhibition has emerged as a promising therapeutic target73. In Chapter
Two, I propose that cardiac p38 inhibition also has the potential to treat cardiac proteinopathies. I demonstrate that inhibition of p38, using p38 inhibitors currently in late stage clinical trials for other diseases, reduce proteotoxicity and restore cardiac function in Bag3P209L Tg+ mice.
Currently, there are no available treatments for Bag3-related cardiomyopathies. To make matters worse, other pre-clinical therapeutics including AAV mediated Bag3 gene therapy and
molecular-chaperone therapies are unlikely to enter the clinic in the near future. AAV mediated gene therapies face numerous obstacles including vector tropism and the cost of treatment188. In
addition, although Bag3 AAV therapy has been utilized in pre-clinical models of
current Bag3 AAV therapies would have an effect, even pre-clinically, in cardiomyopathies resulting from Bag3 mutations. Chaperone-based therapeutics, on the other hand, still have relatively unknown consequences, and have not progressed into the clinic189. Although
promising in theory, questions related to chaperone specificity, side effects, and long term safety still remain. Current molecular chaperone therapeutics, including JG-98 which inhibits the interaction between Hsp70 and Bag3, is still considered a tool compound, which substantial off- target affinities with unknown consequences190,191.
Inhibitors of p38, however, have already faced many of the hurdles described above192.
Latest generation p38 inhibitors have significantly improved specificity and potency over early p38 inhibitors and are safe to use clinically116. As a result, there are currently several Phase II
and Phase III trials of p38 inhibitors that show success. In fact, in a recent clinical trial for Alzheimer’s disease, p38 inhibition has had positive effects on primary patient outcomes
including improved memory and reduced neuronal protein accumulation74,75. Due to the need for
therapeutics, and the preclinical results detailed in this work, future studies in pre-clinical models of Bag3-related cardiomyopathy should focus on proteotoxic p38 inhibition as a promising therapeutic intervention, with the goal of translating p38 inhibitors for clinical use in Bag3- related cardiomyopathies.
Mitochondrial impairment and redox dysfunction in Bag3-related cardiomyopathy
In Chapter Three, I couple transcriptomic and proteomic analyses with ultrastructural analysis of Bag3P209L Tg+ hearts. As expected from our results in Chapter Two, transcripts and
protein levels of PQC components are unchanged in Bag3P209L Tg+ mice, consistent with the
post-translational proteotoxic mechanism described. Interestingly, of the transcript gene sets and proteins that were differentially regulated in Bag3P209L Tg+ mice, a disproportionate number are
mitochondria-associated and involved in metabolic energetics and oxidoreductase activity. Human hearts harboring Bag3 mutations, including the Bag3P209L mutation exhibit cardiac
mitochondrial impairment93. Consistent with this observation, Bag3P209L Tg+ mice also display
mitochondrial abnormalities, including fragmentation and impaired dynamics, leading us to further investigate Bag3-related mitochondrial pathobiology. To determine the cause of mitochondrial impairment, we discovered mitophagy was not impaired in Bag3P209L Tg+ mice
and reactive oxygen species (ROS) were not increased. Surprisingly, however, we also identified dramatic changes in the NAD/NADH and protein acetylation, which both participate in
mitochondrial energetics and redox regulation in disease contexts165,170. While p38 inhibition
seemed to partially restored mitochondrial fragmentation and dynamics, NAD depletion and protein hyperacetylation were unchanged.
The inability of p38 inhibition to restore NAD levels and reduce protein hyperacetylation, even while cardiac dysfunction was completely restored is an intriguing phenomenon that
requires further investigation. In this work, global NAD levels and protein acetylation were identified; however, both NAD and protein acetylation are known to have compartment-specific cellular effects193. Therefore, future studies should start by interrogating which protein
compartment, or compartments are responsible for the observed findings. Additionally, the cardiac effects of modulating NAD levels and protein acetylation should be tested. It is possible that the observed effects are cardioprotective in Bag3P209L related cardiomyopathy. On the other
hand, these changes could just as easily be part of the disease pathology, however they have not yet reached thresholds to have a noticeable effect on cardiac function. Determining the role of NAD depletion and protein hyperacetylation in Bag3P209L Tg+ mice will help us further
acetylation, either through NAD supplementation or antioxidants may complement long term p38 treatment in Bag3-related cardiomyopathy.
Collectively, this work has uncovered important features of Bag3-related
cardiomyopathy, including proteotoxic p38 signaling which drives cardiac dysfunction, as well as mitochondrial abnormalities, NAD depletion and hyperacetylation which may be secondary pathology to proteotoxic disease progression. Additionally, this work has identified p38 inhibition as a potential therapeutic to treat Bag3-related cardiomyopathies, for which no treatments currently exist.