HYPERACETYLATED PROTEINS IN Bag3P209L RELATED CARDIOMYOPATHY
Summary
Mutations in Bcl2 associated athanogene 3 (Bag3) result in cardiac and skeletal muscle myopathies characterized by cardiomyocyte protein accumulation, dissolution of myofibers and progressive muscle wasting, however, our understanding of the underlying molecular
mechanisms leading to the observed pathology remain incomplete. To investigate the etiology of Bag3-related cardiomyopathy, we utilize a cardiac-specific Bag3P209L transgenic (Tg+) model of
Bag3-related cardiomyopathy, that parallels human disease. Bag3P209L Tg+ mice display cardiac
dysfunction, and morphologically, exhibit pre-amyloid oligomers (PAOs) consistent with proteotoxic aggregation and human Bag3-related disease pathology. Previously, we utilized this
Bag3P209L Tg+ model to interrogate cardiac proteotoxic signaling, discovering the p38 signaling
cascade as an important regulator of PAO aggregation and cardiac dysfunction. In this study we continue to examine cellular dysfunction in Bag3P209L Tg+ mice using a combination of
transcriptomic and proteomic analyses to globally assess changes in Bag3P209L Tg+ hearts and
gain further insights into Bag3-related disease pathobiology. Using this approach, we identify differential regulation of only a small fraction of RNA transcripts and proteins in Bag3P209L Tg+
hearts, including disproportionate alterations in mitochondrial related genes and oxidative defense systems. Next, we interrogated Bag3P209L mitochondria, identifying fragmented,
impaired mitochondria that are only partially restored by p38 inhibition. Next, we investigated potential drivers of the observed mitochondrial impairment and identified NAD depletion and
cardiac protein hyperacetylation, but not impaired mitophagy, as potential mechanisms driving mitochondrial dysfunction. Interestingly, p38 inhibitors had no effect on cellular NAD levels or protein hyperacetylation, yet cardiac dysfunction was completely restored by p38 inhibition, suggesting mitochondrial dysfunction, NAD depletion, and protein hyperacetylation are not primary pathologies, but rather secondary phenotypes in Bag3P209L cardiomyopathy.
Introduction
Mutations in Bcl2-associated Athanogene 3 (Bag3) are reported to cause cardiac and skeletal muscle myopathies111. As a small heat shock protein, Bag3 functions to maintain
structural support and PQC processes within muscle cells55. In contrast, mutant Bag3 leads to
progressive cardiac and skeletal muscle weakness. To date, more than 30 mutations in Bag3 have been reported to result in myopathies, many with particularly severe phenotypes affecting
cardiac muscle86,88–90,92,139–141. The most frequently published Bag3 mutation is a P209L
mutation, which results in a severe childhood onset form cardiomyopathy87,139.
To delineate how the Bag3P209L mutation causes cardiomyopathy in patients, we
generated a transgenic mouse model (Bag3P209L Tg+ mice) expressing human Bag3P209L
exclusively in cardiomyocytes101. Bag3P209L Tg+ mice begin to display cardiac dysfunction by
eight months of age, with progressively declining cardiac function through 16 months of age without any mortality. At the cellular level, we identified deficits and remodeling consistent with known hallmarks of human cardiac proteinopathies, including cardiomyocyte enlargement (hypertrophy), PAOs, mitochondrial abnormalities, altered metabolism, Bag3 haploinsufficiency and increased cardiac fibroblast presence. In chapter 2, we focused predominantly on cardiac PAOs and elucidated proteotoxic p38 signaling as a central mechanism leading to cardiac
dysfunction in Bag3P209L Tg+ mice. However, the etiology of proteinopathies are often
multifactorial, leading us to further interrogate cellular dysfunction in Bag3P209L Tg+ hearts.
To our knowledge, Bag3P209L Tg+ mice, are one of only a few animal models of cardiac
proteinopathies, providing us the unique opportunity to investigate cellular dysfunction in a way previously unavailable to investigators142. Although transcriptomic and proteomic analyses have
been widely performed in models of Alzheimer’s, Parkinson’s and Huntington’s disease, no global transcriptomic and proteomic data exists for many cardiac proteinopathies, including Bag3-related cardiomyopathies143–147. Therefore, in the present study, we utilize a combined
transcriptomics and proteomics approach to investigate cellular dysfunction in Bag3P209L Tg+
hearts. Interestingly, our analyses identified differentially expressed mitochondrial-associated gene and protein sets, enriched for redox components, in Bag3P209L Tg+ mice.
Mitochondrial dysfunction is a common pathology in both neurodegenerative and cardiac proteinopathies148. In neurodegenerative proteinopathies, mitochondrial impairment has been
implicated by some as a driver of dysfunction, while others view mitochondrial abnormalities and dysfunction as a secondary effect of disease progression3. In cardiac proteinopathies,
including Bag3-related cardiomyopathy, mitochondrial abnormalities are frequently identified in histopathological analyses, but their role in the pathophysiology of Bag3-related
cardiomyopathies remains unclear149. Bag3 was shown to participate in mitophagy by regulating
parkin66. In a histopathological analysis of a heart from a patient with a Bag3P209L mutation, mild
decreases in mitophagy markers were identified and mitochondrial fragmentation was attributed to decreased mitochondrial clearance93. In other studies, mitochondrial abnormalities in Bag3-
related cardiomyopathies have been linked to oxidative stress and impaired antioxidant defense150. Since, cardiac mitochondrial fragmentation is a known feature in Bag3P209L Tg+
mice, and differential expression of mitochondrial-associated genes and proteins were identified here, we next investigated Bag3P209L related mitochondrial pathophysiology.
In this study, we report that mitochondrial fragmentation and impaired mitochondrial dynamics in Bag3P209L Tg+ mice are not likely the result of impaired mitophagy, but may be the
result of an impaired cellular redox environment. Furthermore, we determine that fragmented mitochondria and mitochondrial dynamics are only partially normalized by p38 inhibitor treatment in Bag3P209L Tg+ mice.
Since cardiac dysfunction is completely restored in Bag3P209L Tg+ mice, this suggests
that either partial restoration of mitochondrial function is sufficient to reverse cardiac
dysfunction or that mitochondrial dysfunction is not a primary pathology in Bag3P209L related
cardiomyopathy. Future studies should focus on the role of mitochondrial energy dynamics, antioxidant defense mechanisms, and protein hyperacetylation on Bag3P209L Tg+ hearts.
Although cardiac function is seemingly not impaired by substantial NAD depletion and protein hyperacetylation, Bag3P209L Tg+ mice may have a weakened response to additional cardiac
insults. Therefore, NAD supplementation or drugs targeting aberrant protein acetylation pathways may have a complementary role in combination with p38 inhibitors to treat Bag3- related cardiomyopathies.
Results
Cardiomyocyte-specific gene expression reveals a potential NAD handling dysfunction in
Bag3P209L Tg+ mice.
Previous studies in our laboratory utilized a cardiac-specific Bag3P209L Tg+ mouse model
to interrogate signaling pathways underlying the disease pathophysiology of cardiac proteinopathies. To further elucidate underlying pathogenic processes, we performed
the control of the aMHC promoter, restricting Bag3P209L expression to cardiomyocytes, we
wanted to determine the cardiomyocyte-specific effects of mutant Bag3P209L in Bag3P209L Tg+
hearts. To do this, we purified and sequenced RNA from isolated cardiomyocytes (CM) and fibroblasts (FB), in parallel, from 12-month-old Bag3P209L Tg+ and age matched sibling Bag3
WT control hearts (Figure 3-1A). We identified a total of 19,094 unique coding genes in CM samples and 19,203 unique coding genes in FB samples. Comparing gene expression in
Bag3P209L Tg+ CMs compared to Bag3 WT CM, we identified 117 differentially expressed genes
utilizing Benjamini-Hochberg adjusted p-values (adj. p < 0.05) from Log2 transformed data
(Figure 1B, left). In FBs from Bag3P209L Tg+ hearts, which do not express mutant Bag3P209L,
only three genes were differentially expressed (adj. p < 0.05), and despite high statistical significance, the Log2 fold change in these three genes was small (Figure 3-1B, right). This
suggests that any effects on fibroblast function in Bag3P209L Tg+ mice are likely post-
transcriptional in nature.
We performed hierarchical cluster analysis on the 120 differentially expressed genes and determined the samples partitioned into two primary groups (Figure 3-1C). The first group was comprised solely of Bag3 WT CM samples. Interestingly, the second group had two unique sub- clusters of samples, the first being Bag3P209L Tg+ CMs and the second being all FB samples.
These data suggest a shift in CM function in Bag3P209L Tg+ hearts. Further supportive of the
causal role that Bag3P209L plays in CMs, the differentially expressed genes associate highly with
dilated cardiomyopathy, cardiomyopathy, and hypertrophic cardiomyopathy when queried using the Jensen disease annotation tool (Figure 3-1D).
Next, we partitioned the genes that were either increased or decreased in Bag3P209L Tg+
PQC transcript levels were unchanged in Bag3P209L cardiomyocytes. Gene annotation enrichment
analysis of the 49 upregulated genes using EnrichR identified gene sets involved in the gamma- tubulin ring complex, trans-Golgi transport vesicles and endocytic vesicles (Figure 3-1E) (Table 3-1). Molecular function analysis of upregulated genes identified an enrichment for genes involved in dehydrogenase activity, oxidoreductase activity, and NADH handling indicative of alterations in metabolism (Figure 3-1E).
Interestingly, of the 68 downregulated proteins in Bag3P209L Tg+ cardiomyocytes,
mitochondria-associated gene sets were among the most enriched, likely indicative of
mitochondrial dysfunction, a known feature of many cardiac myopathies, including Bag3-related cardiac myopathy (Figure 3-1F). However, the precise mechanisms leading to mitochondrial dysfunction in Bag3-related cardiomyopathy remain to be elucidated. Downregulated proteins were also enriched for myosin filament and myofibril proteins, which is unsurprising as
cardiomyopathy is associated with myofilament disruption and wasting153,154. Furthermore, gene
annotation analysis for molecular function identified genes largely involved in muscle actin binding, which when disrupted, can lead to cardiomyopathy155,156.
Whole ventricle proteomics on Bag3P209L Tg+ hearts reveals an upregulation in endocytic protein expression and downregulation of mitochondrial oxidative defense.
In a small proteomics study, proteins were extracted from the whole heart of 16-month- old sibling Bag3P209L Tg+ and Bag3 WT mice and sequenced by MS/MS (Figure 3-2A). After
filtering to exclude proteins with fewer than 2 identified peptides, a total of 494 proteins were identified by MS/MS analysis, of which 67 were differentially regulated (>50% change in expression) between Bag3P209L Tg+ and Bag3 WT hearts, including 18 downregulated and 49
PQC transcript level changes and our previous report that protein levels of several major PQC protein are largely unchanged in Bag3P209L induced pathology. However, analysis of altered
proteins in Bag3P209L Tg+ mice do point to an upregulation of endocytic vesicle assembly,
essential in proteotoxic stress response pathways including autophagy and aggrephagy62.
Additionally, upregulated protein sets are enriched for proteins involved in redox systems, lipid binding, and NAD handling (Figure 3-2E).
Correlating with the identification of mitochondrial impairment, and the enrichment for redox components identified in our transcriptomic analysis, mitochondrial associated proteins make up the largest group of downregulated proteins in Bag3P209L Tg+ mice. Of the total 494
identified proteins, 24% are mitochondria-associated (Figure 3-2C); however, 67% of downregulated proteins in Bag3P209L Tg+ mice are mitochondrial, and 27% of upregulated
proteins (Figure 3-2D). Molecular functional annotation further identified downregulated proteins were enriched for mitochondrial oxidative defense and NAD handling proteins (Figure 3-2F). Improper NAD handling, NAD+ depletion, and reactive oxygen species are hallmarks of mitochondrial impairment, and have been linked to PQC disruption and cardiomyopathy157.
Although mitochondrial deficits have been reported in patients with Bag3-related myopathies, and other protein-aggregation myofibrillar myopathies, it has largely been attributed as a
secondary effect in the disease pathophysiology and few studies have focused on the dysfunction in depth. However, due to the importance of mitochondrial function in the heart, the role of mitochondria in disease progression in other proteinopathies, and increased interest in targeting mitochondria therapeutically we sought to further understand mitochondrial dysfunction in
Bag3P209L Tg+ mice.
Bag3P209L Tg+ mice display mitochondrial abnormalities that are only partially restored by
Since both cardiomyocyte transcriptomics and whole ventricle proteomics identified significant downregulation of mitochondrial components, we hypothesized that mitochondrial dysfunction is a central pathophysiological feature in Bag3P209L Tg+ mice. To test our
hypothesis, we first used TEM to look for any changes in the ultrastructure of cardiac
mitochondria from Bag3P209L Tg+ compared to Bag3 WT control mice. Indeed, TEM imaging of
Bag3P209L Tg+ cardiomyocytes compared to Bag3 WT controls indicated some degree of
morphological impairment of Bag3P209L Tg+ mitochondria, with the presence of smaller
fragmented mitochondria in Bag3P209L Tg+ cardiomyocytes (Figure 3-3A). We confirmed this
change quantitatively, measuring mitochondria number and mitochondrial area. Bag3P209L Tg+
hearts have an increase in mitochondrial number and a decrease in total mitochondrial area compared to control hearts (Figure 3-3, B and C). This increase in fragmented mitochondria was further supported by qPCR of mitochondrial DNA, which show an increase in two markers of mitochondrial DNA (ND1 and CO1) (Figure 3-3, D-F). In chapter 2, we showed that p38/MAPK inhibition restored cardiac function by 10 days of treatment and reduced proteotoxicity
contributing to disease progression. Since p38 treatment successfully restored cardiac function, we hypothesized that p38 inhibition would have a positive impact on Bag3P209L mitochondrial
biogenesis. Surprisingly, only a partial recovery of mitochondria number and area was observed, suggesting that mitochondrial impairment is independent of aberrant p38 signaling and 10-day treatment with p38 inhibitors is insufficient to normalize mitochondrial morphology (Figure 3-3). Mitochondrial fusion and fission processes are disrupted in Bag3P209L Tg+ hearts
Fusion and fission events regulate dynamic mitochondrial morphological changes in response to both metabolic or environmental stresses158. Under physiological conditions, fusion
normal mitochondria function. Fission, allows healthy mitochondria to create new mitochondria as needed, and damaged mitochondria to fragment, facilitating mitochondrial degradation. Under pathological stress, however, disruptions in fusion and fission dynamics can result in
mitochondrial fragmentation159. In fact, mitochondrial fragmentation has been implicated in
numerous proteinopathies including Alzheimer’s and Parkinson’s disease148. The presence of
smaller, fragmented mitochondria in Bag3P209L Tg+ hearts led us to believe that the regulation of
mitochondrial fission and fusion may be disrupted. Mitochondrial fusion is largely regulated by mitofusion (mfn1) and optic atrophy (opa1) while mitochondrial fission events are regulated by dystrophin-related protein 1 (drp1) and Fission 1 (Fis1) (Figure 3-4A). By qPCR we identified significant decreases in mRNA in Bag3P209L Tg+ mice from both major mitochondrial fusion
genes mfn1 (Figure 3-4B) and opa 1 (Figure 4C). We also identified a decrease in mitochondrial fission gene drp1 (Figure 3-4D), while a mild decrease in fis1 was not statistically significant (Figure 3-4E). Collectively, this data indicates an impairment of mitochondrial dynamics that may explain the increased fragmented mitochondria in Bag3P209L Tg+ mice. Interestingly, p38
inhibitor treated Bag3P209L Tg+ mice no longer have significantly decreased mfn1, opa1, and
drp1 indicative of a partial recovery in mitochondrial dynamics consistent with the partial recovery demonstrated in mitochondrial number and area.
Mitophagy is not dysregulated in Bag3P209L Tg+ hearts.
In a previous study, we reported that Bag3P209L Tg+ hearts did not have global changes in
autophagic flux101. However, our analysis did not preclude the possibility that specific
autophagic processes could be dysregulated. Mitophagy is a specialized autophagic process responsible for the degradation of entire dysfunctional mitochondria in response to both physiological and pathological cellular stress events where mitochondria is damaged beyond
repair160. Fragmented mitochondria are often targeted for mitophagy and when mitophagy is
disrupted, a buildup of fragmented dysfunctional mitochondria can occur. To probe mitophagy, we immunoblotted for expression levels of parkin and pink1, known markers of mitophagy that increase significantly upon mitophagy initiation (Figure 3-5, A-C). However, neither Parkin, nor Pink 1 were significantly increased in Bag3P209L Tg+ hearts, suggesting that dysregulated
mitophagy is not responsible for the observed mitochondrial phenotype.
Bag3P209L Tg+ mice have depleted cardiac NAD+
Mitochondrial dysfunction in proteinopathies is commonly linked to an increase in toxic reactive oxygen species (ROS)161,162. Although the mechanisms are incompletely understood,
PQC impairment and proteotoxic signaling has been shown to result in transient increases in cytoplasmic and mitochondrial ROS. Increases in cellular ROS lead to oxidative stress resulting in DNA damage, inflammation, apoptosis, and mitochondrial damage including fragmentation and impaired mitochondrial dynamics163,164. Differentially regulated gene sets from our
transcriptomic and proteomic datasets display an enrichment for redox components, suggesting a potential impairment of oxidative defenses in Bag3P209L Tg+ mice. Using electron paramagnetic
resonance, we analyzed ROS directly and quantitatively in hearts isolated from Bag3P209L Tg+
and Bag3 WT mice. Surprisingly, there was no change in ROS levels in Bag3P209L Tg+ hearts
compared to control Bag3 WT mice (Figure 3-6A). While transient ROS species can be difficult to detect for a variety of biological reasons, technical limitations, cellular oxidative stress can also be probed by investigating the NAD+/NADH redox couple. NAD+/NADH is a major regulator of cellular energy metabolism and changes in NAD+/NADH handling are recognized as a crucial indicator of oxidative stress165. Furthermore, much like ROS, deficiency and
proteinopathies and metabolic disorders157,166. Remarkably, we discovered that Bag3P209L Tg+
hearts have a 50% decrease in the NAD/NADH ratio, indicative of substantial oxidative and metabolic dysfunction (Figure 6B). The NAD pool is essential for mitochondrial function, and NAD deficiency can result in mitochondrial dysfunction and fragmentation167. Furthermore,
decreased NAD can also impact protein acetylation, an important post-translational modification implicated in a myriad of cell signaling pathways.
Bag3P209L Tg+ mice have increased cardiac protein acetylation
A potential consequence of decreased cardiac pools of NAD is a pathologic increase in protein acetylation. Recent studies have shown that decreased cardiac NAD can cause the
hyperacetylation of mitochondrial proteins resulting in damaged, dysfunctional mitochondria and impaired response to cardiac stress168. Linking NAD and acetylation are Sirtuins (SIRT), a
family of NAD dependent deacetylases responsible for protein deacetylation169. Decreased SIRT
activity has been shown to result in protein hyperacetylation under pathologic stress in a number of metabolic diseases170. Strikingly, immunoblots for acetylated lysine demonstrate a > 7.0-fold
increase in total protein acetylation in Bag3P209L Tg+ mice compared to Bag3 WT controls
(Figure 3-7, A and B). However, testing for universal SIRT activity, we detected no change in total SIRT activity (Figure 3-8). Proteomic analysis detected a downregulation in mitochondrial expressed SIRT5 suggesting that altered activity in either specific SIRT family members, or within specific compartments of cardiomyocytes, may result the dramatic increase in protein acetylation observed.
NAD+ depletion and aberrant protein acetylation persist in aged Bag3P209L Tg+ mice after p38 MAPK inhibition.
In Chapter Two, we demonstrated that p38 inhibition reduces cardiac proteotoxicity and restores cardiac function in Bag3P209L Tg+ mice. To determine if p38 inhibition was able to
restore NAD/NADH levels and reduce protein acetylation, we tested p38 inhibitor treated hearts. Intriguingly, p38 inhibition had no effect on either NAD levels (Figure 3-6B) or total protein acetylation (Figure 3-7, A and B). Collectively this data suggests that while p38 treatment is able to reduce proteotoxicity and restore cardiac function, some Bag3P209L induced impairment exists
following 10 days of p38 inhibitor treatment. Importantly, however, p38 inhibition does not