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. 2020 Aug 13:7:142.
doi: 10.3389/fcvm.2020.00142. eCollection 2020.

Association of Genetic Variants at TRPC6 With Chemotherapy-Related Heart Failure

Nadine Norton  1 , Julia E Crook  2 , Liwei Wang  3 , Janet E Olson  3 , Jennifer M Kachergus  1 , Daniel J Serie  2 , Brian M Necela  1 , Paul G Borgman  1 , Pooja P Advani  4 , Jordan C Ray  5 , Carolyn Landolfo  5 , Damian N Di Florio  5   6 , Anneliese R Hill  5 , Katelyn A Bruno  5   6 , DeLisa Fairweather  5   6
Affiliations

Association of Genetic Variants at TRPC6 With Chemotherapy-Related Heart Failure

Nadine Norton et al. Front Cardiovasc Med. .

Abstract

Background: Our previous GWAS identified genetic variants at six novel loci that were associated with a decline in left ventricular ejection fraction (LVEF), p < 1 × 10-5 in 1,191 early breast cancer patients from the N9831 clinical trial of chemotherapy plus trastuzumab. In this study we sought replication of these loci. Methods: We tested the top loci from the GWAS for association with chemotherapy-related heart failure (CRHF) using 26 CRHF cases from N9831 and 984 patients from the Mayo Clinic Biobank which included CRHF cases (N = 12) and control groups of patients treated with anthracycline +/- trastuzumab without HF (N = 282) and patients with HF that were never treated with anthracycline or trastuzumab (N = 690). We further examined associated loci in the context of gene expression and rare coding variants using a TWAS approach in heart left ventricle and Sanger sequencing, respectively. Doxorubicin-induced apoptosis and cardiomyopathy was modeled in human iPSC-derived cardiomyocytes and endothelial cells and a mouse model, respectively, that were pre-treated with GsMTx-4, an inhibitor of TRPC6. Results: TRPC6 5' flanking variant rs57242572-T was significantly more frequent in cases compared to controls, p = 0.031, and rs61918162-T showed a trend for association, p = 0.065. The rs61918162 T-allele was associated with higher TRPC6 expression in the heart left ventricle. We identified a single TRPC6 rare missense variant (rs767086724, N338S, prevalence 0.0025% in GnomAD) in one of 38 patients (2.6%) with CRHF. Pre-treatment of cardiomyocytes and endothelial cells with GsMTx4 significantly reduced doxorubicin-induced apoptosis. Similarly, mice treated with GsMTx4 had significantly improved doxorubicin-induced cardiac dysfunction. Conclusions: Genetic variants that are associated with increased TRPC6 expression in the heart and rare TRPC6 missense variants may be clinically useful as risk factors for CRHF. GsMTx-4 may be a cardioprotective agent in patients with TRPC6 risk variants. Replication of the genetic associations in larger well-characterized samples and functional studies are required.

Keywords: GsMTx-4; TWAS; anthracycline; breast cancer; cardiomyopathy; cardiotoxicity; doxorubicin; trastuzumab.

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Figures

Figure 1
Figure 1
Flow diagram of patient populations and experimental plan. QC, quality control; PCA, principal component analysis; GWAS, genome-wide association study; LVEF, left ventricular ejection fraction; CHF, congestive heart failure, HF, heart failure.
Figure 2
Figure 2
Imputed TRPC6 gene expression levels in heart left ventricle are associated with decline in LVEF in breast cancer patients who were treated with anthracycline and trastuzumab. PrediXcan was used to impute gene expression in left ventricular cardiac tissue of patients from N9831 and test for association analysis of gene expression with maximum decline in LVEF. Gene expression was imputed for 4,853 genes. TRPC6 (one of the top hits in the primary GWAS analysis) ranked 23/4,853 genes.
Figure 3
Figure 3
TRPC6 exon sequencing identified a rare missense variant in a breast cancer patient with chemotherapy-related heart failure. rs767086724 Asn338Ser (heterozygote) identified in 32 year old female patient (N9831 clinical trial, Arm B: sequential doxorubicin + trastuzumab) with chemotherapy-induced HF.
Figure 4
Figure 4
GsMTx-4 reduces doxorubicin-induced apoptosis. Apoptosis (luminescence units) in doxorubicin (Dox)-treated cells +/– GsMTx4 (5 μM) were compared by unpaired T-test in iPSC-derived cardiomyocytes (A) and iPSC-derived endothelial cells (B). Error bars are standard deviation of the mean.
Figure 5
Figure 5
Doxorubicin causes fibrosis and cardiomyopathy that is prevented by Trpc6 inhibition. Male B6.129 WT mice were injected intraperitoneally (ip) with doxorubicin (Dox) on days 1, 3, 5, 8, 10, 12 (cumulative dose 24 mg/kg) ± the Trpc6 inhibitor GsMTx-4 (10 mg/kg) or saline/DMSO control (Con) ip during Dox therapy and echocardiography-derived LVEF and global longitudinal strain (GLS) performed at day 21. (A) Dox-induced fibrosis was prevented in mice that were pre-treated with GsMTx-4. 1-Way ANOVA test revealed significant differences between the 3 groups, p = 0.0005. (B) Dox-induced decrease in LVEF was prevented in mice that were pre-treated with GsMTx-4. 1-Way ANOVA test revealed significant differences between the 3 groups, p = 0.0004. (C) GLS measures of mice in Con and Dox-treated groups were comparable to normal function and impaired cardiac function in humans, respectively. Dox-induced increase in GLS was prevented in mice that were pre-treated with GsMTx4. 1-Way Kruskal-Wallace test revealed significant differences between the 3 groups, p = 0.0006. (D) Parametric maps of radial strain of the endocardium through 3 cardiac cycles. The larger spread of the lines during systole in a representative wild type Dox male mouse depicts less coordinated contraction (i.e., cardiac dysfunction) compared to a wild type Con mouse. Data are shown as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001 using unpaired test. Multiple-comparisons of groups was performed using Mann-Whitney or Sidak tests with a Bonferroni adjusted p-value for 3 independent tests of <0.017 regarded as significant.
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