Protein Turnover and Quality Control of Cardiac Myosin Binding Protein C in Hypertrophic Cardiomyopathy

The cardiac sarcomere is a complex matrix of molecular machinery responsible for contraction of the heart. Proper contractile function relies on maintenance of rigid stoichiometry of myofilament proteins and multiple protein quality control (PQC) pathways. Though PQC is undeniably essential to sarcomere protein homeostasis (proteostasis), there are many gaps in our knowledge of factors that regulate turnover of sarcomere proteins. Mutations in many of these proteins cause hypertrophic cardiomyopathy (HCM), which is often designated a “sarcomeropathy.” HCM and is characterized by thickening of the left ventricular free wall and intraventricular septum, myocardial fibrosis, and diastolic dysfunction. The most commonly mutated gene in HCM is cardiac myosin binding protein C (MYBPC3). The primary pathogenic mechanisms associated with MYBPC3 mutations remain unresolved. PQC and protein turnover are central to two hypotheses with the most supportive evidence: haploinsufficiency of MYBPC3 in the sarcomere, and proteotoxicity of truncated MYBPC3 protein products. However, very little is known about how MYBPC3 interacts with PQC networks in either physiological or pathological conditions. The goal of this thesis was to investigate pathogenic mechanisms associated with different MYBPC3 mutations, explore the roles of protein homeostasis and turnover with respect to these mechanisms, and identify PQC factors which are involved in MYBPC3 turnover. Using affinity purification-mass spectrometry, we identified several molecular chaperones as potential novel interactors with MYBPC3, including αB-crystallin and HSP27, and the inducible and constitutive isoforms of the ubiquitous heat shock protein 70kDa family (HSP70 and HSC70, respectively). We then confirmed that HSP70 chaperones regulate proteasomal degradation of MYBPC3 by modulating their expression and activity and observing effects on MYBPC3 protein half-life in a primary cardiomyocyte culture system. This represents to our knowledge the first identification of a chaperone associated with MYBPC3. To determine the extent to which proteotoxicity of truncated MYBPC3 contributes to HCM pathogenesis in isolation from haploinsufficiency, we explored the effects of acute and chronic expression of truncated MYBPC3 on cardiomyocyte PQC using primary cell culture and a transgenic mouse model, respectively. We reported no deleterious effects of truncated MYBPC3 expression on proteostasis in vitro or in vivo. Further, chronic expression of a truncating MYBPC3 transgene in mice up to 12 months of age was not sufficient to elicit hypertrophic remodeling. These results challenge the hypothesis that truncated MYBPC3 is directly proteotoxic and suggest a “poison peptide” mechanism may not be relevant to HCM pathogenesis without concurrent MYBPC3 haploinsufficiency. Lastly, we investigated the locus-dependency of protein stability in non-truncating MYBPC3 mutations. Novel analysis of genotypes using the Sarcomeric Human Cardiomyopathy Registry (SHaRe) of HCM patients uncovered putative clusters of missense mutations in the C3, C6 and C10 domains of MYBPC3. We identified a consistent pattern in C10 mutants of lack of sarcomere incorporation and markedly rapid degradation. This was in contrast to C3 and C6 mutants, which were generally equally stable as WT MYBPC3 and localized correctly within the sarcomere. These findings demonstrate mutation locus significantly influences protein stability and turnover, and further dissect pathogenic mechanisms associated with non-truncating mutations. With these studies, we have moved toward clarifying pathogenic mechanisms in MYBPC3-linked HCM, which will inform future development of targeted interventions for patients with different genotypes. Furthermore, we have identified new potential therapeutic targets to restore normal stoichiometry to haploinsufficient sarcomeres, and contributed to our understanding of the enigmatic process of sarcomere protein quality control.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/149802/1/glaziera_1.pd

Spatial and Functional Distribution of MYBPC3 Pathogenic Variants and Clinical Outcomes in Patients with Hypertrophic Cardiomyopathy

Background - Pathogenic variants in MYBPC3, encoding cardiac MyBP-C, are the most common cause of familial hypertrophic cardiomyopathy. A large number of unique MYBPC3 variants and relatively small genotyped HCM cohorts have precluded detailed genotype-phenotype correlations. Methods - Patients with HCM and MYBPC3 variants were identified from the Sarcomeric Human Cardiomyopathy Registry (SHaRe). Variant types and locations were analyzed, morphologic severity was assessed, and time-event analysis was performed (composite clinical outcome of sudden death, class III/IV heart failure, LVAD/transplant, atrial fibrillation). For selected missense variants falling in enriched domains, myofilament localization and degradation rates were measured in vitro. Results - Among 4,756 genotyped HCM patients in SHaRe, 1,316 patients were identified with adjudicated pathogenic truncating (N=234 unique variants, 1047 patients) or non-truncating (N=22 unique variants, 191 patients) variants in MYBPC3. Truncating variants were evenly dispersed throughout the gene, and hypertrophy severity and outcomes were not associated with variant location (grouped by 5' - 3' quartiles or by founder variant subgroup). Non-truncating pathogenic variants clustered in the C3, C6, and C10 domains (18 of 22, 82%, p<0.001 vs. gnomAD common variants) and were associated with similar hypertrophy severity and adverse event rates as observed with truncating variants. MyBP-C with variants in the C3, C6, and C10 domains was expressed in rat ventricular myocytes. C10 mutant MyBP-C failed to incorporate into myofilaments and degradation rates were accelerated by ~90%, while C3 and C6 mutant MyBP-C incorporated normally with degradation rate similar to wild-type. Conclusions - Truncating variants account for 91% of MYBPC3 pathogenic variants and cause similar clinical severity and outcomes regardless of location, consistent with locus-independent loss-of-function. Non-truncating MYBPC3 pathogenic variants are regionally clustered, and a subset also cause loss-of-function through failure of myofilament incorporation and rapid degradation. Cardiac morphology and clinical outcomes are similar in patients with truncating vs. non-truncating variants

CRISPR/Cas9-Mediated Constitutive Loss of VCP (Valosin-Containing Protein) Impairs Proteostasis and Leads to Defective Striated Muscle Structure and Function In Vivo

Valosin-containing protein (VCP) acts as a key regulator of cellular protein homeostasis by coordinating protein turnover and quality control. Mutations in VCP lead to (cardio-)myopathy and neurodegenerative diseases such as inclusion body myopathy with Paget’s disease of the bone and frontotemporal dementia (IBMPFD) or amyotrophic lateral sclerosis (ALS). To date, due to embryonic lethality, no constitutive VCP knockout animal model exists. Here, we generated a constitutive CRISPR/Cas9-induced vcp knockout zebrafish model. Similar to the phenotype of vcp morphant knockdown zebrafish embryos, we found that vcp-null embryos displayed significantly impaired cardiac and skeletal muscle function. By ultrastructural analysis of skeletal muscle cells and cardiomyocytes, we observed severely disrupted myofibrillar organization and accumulation of inclusion bodies as well as mitochondrial degeneration. vcp knockout was associated with a significant accumulation of ubiquitinated proteins, suggesting impaired proteasomal function. Additionally, markers of unfolded protein response (UPR)/ER-stress and autophagy-related mTOR signaling were elevated in vcp-deficient embryos, demonstrating impaired proteostasis in VCP-null zebrafish. In conclusion, our findings demonstrate the successful generation of a stable constitutive vcp knockout zebrafish line that will enable characterization of the detailed mechanistic underpinnings of vcp loss, particularly the impact of disturbed protein homeostasis on organ development and function in vivo

HSC70 is a chaperone for wild-type and mutant cardiac myosin binding protein C

Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover

Depressive Disorders During Pregnancy

OBJECTIVE: To estimate the prevalence of major and minor depression, panic disorder, and suicidal ideation during pregnancy while also identifying factors independently associated with antenatal depressive disorders. METHODS: In this prospective study, participants were 1,888 women receiving ongoing prenatal care at a university obstetric clinic from January 2004 through January 2009. Prevalence of psychiatric disorders was measured using the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria based on the Patient Health Questionnaire. Multiple logistic regression identified factors associated with probable major depressive disorder and any depressive disorder. RESULTS: Antenatal depressive disorders were present in 9.9% with 5.1% (97) meeting criteria for probable major depression and 4.8% (90) meeting criteria for probable minor depression. Panic disorder was present in 3.2% (61), and current suicidal ideation was reported by 2.6% (49). Among patients with probable major depression, 29.5% (28) reported current suicidal ideation. Psychosocial stress (odds ratio [OR], 1.29; 95% confidence interval [CI], 1.21–1.36), domestic violence (OR 3.45; 95% CI 1.46–8.12), chronic medical conditions (OR 3.05; 95% CI 1.63–5.69), and race (Asian: OR 5.81; 95% CI 2.55–13.23; or African American: OR 2.98; 95% CI 1.24–7.18) each significantly increased the odds of probable antepartum major depressive disorder, whereas older age (OR 0.92; 95% CI 0.88–0.97) decreased the odds. Factors associated with odds of any depression were similar overall except that Hispanic ethnicity (OR 2.50; 95% CI 1.09–5.72) also independently increased the odds of any depression. CONCLUSION: Antenatal major and minor depressive disorders are common and significantly associated with clinically relevant and identifiable risk factors. By understanding the high point prevalence and associated factors, clinicians can potentially improve the diagnosis and treatment rates of serious depressive disorders in pregnant women