Association of Variants in BAG3 With Cardiomyopathy Outcomes in African American Individuals

Abstract

Importance: The prevalence of nonischemic dilated cardiomyopathy (DCM) is greater in individuals of African ancestry than in individuals of European ancestry. However, little is known about whether the difference in prevalence or outcomes is associated with functional genetic variants.

Objective: We hypothesized that Bcl2-associated anthanogene 3 (BAG3) genetic variants were associated with outcomes in individuals of African ancestry with DCM.

Design: This multicohort study of the BAG3 genotype in patients of African ancestry with dilated cardiomyopathy uses DNA obtained from African American individuals enrolled in 3 clinical studies: the Genetic Risk Assessment of African Americans With Heart Failure (GRAHF) study; the Intervention in Myocarditis and Acute Cardiomyopathy Trial-2 (IMAC-2) study; and the Genetic Risk Assessment of Cardiac Events (GRACE) study. Samples of DNA were also acquired from the left ventricular myocardium of patients of African ancestry who underwent heart transplant at the University of Colorado and University of Pittsburgh.

Main outcomes and measures: The primary end points were the prevalence of BAG3 mutations in African American individuals and event-free survival in participants harboring functional BAG3 mutations.

Results: Four BAG3 genetic variants were identified; these were expressed in 42 of 402 African American individuals (10.4%) with nonischemic heart failure and 9 of 107 African American individuals (8.4%) with ischemic heart failure but were not present in a reference population of European ancestry (P < .001). The variants included 2 nonsynonymous single-nucleotide variants; 1 three-nucleotide in-frame insertion; and 2 single-nucleotide variants that were linked in cis. The presence of BAG3 variants was associated with a nearly 2-fold (hazard ratio, 1.97 [95% CI, 1.19-3.24]; P = .01) increase in cardiac events in carriers compared with noncarriers. Transfection of transformed adult human ventricular myocytes with plasmids expressing the 4 variants demonstrated that each variant caused an increase in apoptosis and a decrease in autophagy when samples were subjected to the stress of hypoxia-reoxygenation.

Conclusions and relevance: This study demonstrates that genetic variants in BAG3 found almost exclusively in individuals of African ancestry were not causative of disease but were associated with a negative outcome in patients with a dilated cardiomyopathy through modulation of the function of BAG3. The results emphasize the importance of biological differences in causing phenotypic variance across diverse patient populations, the need to include diverse populations in genetic cohorts, and the importance of determining the pathogenicity of genetic variants.

Figure 1.. Kaplan-Meier Curves Showing Event-Free Survival in Patients With or Without a BAG3 Genetic Variant

Kaplan-Meier curves show event-free survival in all patients (A), patients with nonischemic heart failure (B), and patients with idiopathic dilated cardiomyopathy (C), with or without a BAG3 variant. GRACE indicates the Genetic Risk Assessment of Cardiac Events trial; GRAHF, the Genetic Risk Assessment of African Americans With Heart Failure trial; IMAC-2, Myocarditis and Acute Cardiomyopathy Trial-2.

Figure 2.. Levels of BAG3 Protein and Messenger RNA (mRNA) in Failing Human Hearts

A, A representative Western blot of protein isolated from human hearts with severe left ventricular dysfunction secondary to idiopathic dilated cardiomyopathy (IDC; n = 23) or ischemic heart disease (n = 16), contrasted with control participants with nonfailing hearts (n = 4). Samples obtained from hearts that were found to carry a BAG3 variant are indicated by the red squares. The unlabeled lanes represent molecular weight markers. B, Quantification of multiple Western blots. C, Quantification of quantitative polymerase chain reaction assessment of mRNA levels in a subset of the same hearts (non-IDC, n = 13; IDC, n = 18; and nonfailing human hearts from control participants, n = 4). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control for both the Western blot and the quantitative polymerase chain reaction.

Figure 3.. Confocal Images of Autophagy Flux and Apoptosis in AC16 Cells

Representative confocal images from AC16 cells that were transfected with an empty plasmid or one containing wild-type BAG3 or c.187C→G+c.1138C→T (p.Pro63Ala + p.Pro380Ser), c.249C→A (p.His83Gln), c.474_476dupGGC (p.Ala160dup), or c.1436C→T (p.Ala479Val) and then cotransfected with adenovirus-red fluorescent protein-green fluorescent protein-microtubule–-associated protein 1A/1B light chain 3. A, Red puncta represent increased LC3 in autophagolysosomes in which green fluorescent protein has been quenched by the increased acidity after lysosomal-autophagasome fusion. B, Hypoxia-reoxygenation increased autophagy in cells with empty vectors or wild-type BAG3, as seen by an increase in yellow-red LC3 puncta; expression of any BAG3 variants resulted in impaired autophagy response and a diminished increase in total LC3 puncta compared with wild-type BAG3. C, A representative confocal image of AC16 cells stained with Annexin-V (green) and propidium iodide (red) to distinguish apoptotic cells (green), late apoptotic and necrotic cells (red), and nonviable cells (green and red). Few apoptotic cells are seen in AC16 cells transfected with wild-type BAG3; there was a marked increase in apoptotic cells in the presence of an empty vector or any BAG3 variant.

Figure 4.. Hemodynamic Effects of Infection With rAAV9-BAG3 vs rAAV9-BAG3^p.P63A+P380S in Mice With BAG3 Haploinsufficiency (cBAG3^+/−) or in Nontransgenic Wild-Type Mice (cBAG3^+/+)

cBAG3^+/− mice aged 4 to 6 weeks received either a retro-orbital injection of 1x10¹² particles of adeno-associated virus 9 (rAAV9)–BAG3^p.P63A+P380S, rAAV9-green fluorescence protein (GFP) or rAAV9-BAG3^wild-type and were then given echocardiograms every other week. In the same experiment, 6-week-old cBAG3^+/+ mice received an injection of either rAAV9-BAG3^p.P63A+P380S, rAAV9-GFP, or rAAV9-BAG3^wild-type. After 6 weeks, left ventricular myocardium was harvested for subsequent analysis. A, A comparison of serial measures of left ventricular ejection fraction by echocardiogram in mice infected with the rAAV9-BAG3^p.P63A+P380S (cBAG3^+/+: n = 4; cBAG3^+/−: n = 3) with mice infected with rAAV9-GFP (cBAG3^+/+: n = 8; cBAG3^+/−: n = 4) shows statistical significant differences at 2, 4, and 6 weeks (P = .01). B, A comparison of mice infected with the rAAV9-BAG3^p.P63A+P380S (cBAG3^+/+: n = 4; cBAG3^+/−: n = 3) with mice expressing rAAV9-BAG3^wild-type (cBAG3^+/+: n = 4; cBAG3^+/−: n = 5) shows statistically significant differences at 4 and 6 weeks (P = .05).