Chemogenetic generation of hydrogen peroxide in the heart induces severe cardiac dysfunction

Abstract

Oxidative stress plays an important role in the pathogenesis of many disease states. In the heart, reactive oxygen species are linked with cardiac ischemia/reperfusion injury, hypertrophy, and heart failure. While this correlation between ROS and cardiac pathology has been observed in multiple models of heart failure, the independent role of hydrogen peroxide (H₂O₂) in vitro and in vivo is unclear, owing to a lack of tools for precise manipulation of intracellular redox state. Here we apply a chemogenetic system based on a yeast D-amino acid oxidase to show that chronic generation of H₂O₂ in the heart induces a dilated cardiomyopathy with significant systolic dysfunction. We anticipate that chemogenetic approaches will enable future studies of in vivo H₂O₂ signaling not only in the heart, but also in the many other organ systems where the relationship between redox events and physiology remains unclear.

Fig. 1

Expression and activation of the HyPer-DAAO fusion protein in cardiac myocytes. a Representative real-time ratiometric fluorescent images of cardiac myocytes isolated from rats infected with an AAV9 construct expressing the HyPer-DAAO fusion protein (DAAO). 4 weeks after infection, isolated myocytes were treated with 10 mM D- or L-alanine for the indicated times, and the HyPer ratio was quantitated. Images are displayed with a lookup table in which the color maps to the ratios shown in the colorbar, and the luminance maps to the intensity of HyPer’s 420 nm excitation. Additional representative images of myocytes treated with D- vs. L-alanine can be found in Supplementary Figure 2 Scale bar, 50 μm. b Pooled ratiometric data from multiple myocytes treated with 5 mM D-alanine (blue circles, n = 32 cells) or L-alanine (red squares, n = 21 cells) for 90 min. Ratios are normalized to the HyPer ratio prior at time t = 0. ***p < 0.001 by two-way ANOVA with Bonferroni correction for multiple comparisons for D- vs. L-alanine. c Immunoblot probed for DAAO expression in lysates prepared from tissues isolated from rats infected with AAV9-DAAO. Lysates were probed with an antibody directed against YFP, which recognizes the HyPer component of the HyPer-DAAO fusion construct. This blot is representative of three experiments that yielded similar results. Data are represented as mean ± standard error

Fig. 2

Fluorescence time course of DAAO activation and in vitro transcriptional responses. a Pooled ratiometric fluorescent data from myocytes treated with 1 mM (green triangles, n = 20 cells), 5 mM (red squares, n = 20 cells), or 10 mM (blue circles, n = 9 cells) D- alanine or 10 mM L-alanine (purple inverted triangles, n = 14 cells) for 45 min. Ratios are normalized to the HyPer ratio prior at time t = 0. **p < 0.01 by two-way ANOVA with Bonferroni correction for multiple comparisons for L- vs. D-alanine. b Ratiometric fluorescence time courses of human IPS-derived cardiac myocytes expressing HyPer-DAAO (blue circles, n = 9 cells) or SypHer2-DAAO (pH control construct; red squares, n = 13 cells) treated with D-alanine (10 mM). ***p < 0.001 by two-way ANOVA with Bonferroni correction for multiple comparisons. c Changes in expression of the Nrf2 transcriptional targets Hmox1, Nqo1, and Sxn1 and the NF-κB transcriptional targets Il1b, Tnfa, Icam1, and Nos2 in cardiac myocytes isolated from rats expressing DAAO and then treated with 10 mM D- vs. L-alanine for 120 min. Distributions for control samples treated with L-alanine can be found on Supplementary Figure 3A. d Relative changes in expression of the redox-active enzymes Prdx1, Prdx2, Prdx3, Gpx1, Gpx3, Txn1, and Txn2 in response to 10 mM D- vs. L-alanine, analyzed in cardiac myocytes isolated from rats infected with DAAO. Distributions for control samples treated with L-alanine can be found on Supplementary Figure 3B. *p < 0.05, **p < 0.01, and ***p < 0.001 D- vs. L-alanine by ANOVA. Data are represented as mean ± standard error

Fig. 3

Echocardiographic and intracardiac pressure parameters after chronic activation of DAAO. a Representative M-mode images from short-axis views of the left ventricle of rats infected with control (Ctrl) or DAAO virus and treated with D-alanine in their drinking water for 4 weeks. b Left ventricular echocardiographic parameters over the 4 weeks of oral D-alanine treatment in rats expressing DAAO (red squares) vs. control animals (blue circles). *p < 0.05, **p < 0.01, and ***p < 0.001 by ANOVA. c Representative left ventricular (LV) pressure traces of Ctrl and DAAO-infected rats after 4 weeks of oral D-alanine treatment. d Pooled LV pressure parameters after 4 weeks of treatment. *p < 0.05 by Mann–Whitney U test. Data are represented as mean ± standard error

Fig. 4

Effects of chronic in vivo DAAO activation on ex vivo cardiac responses and signaling. a Representative traces of tension generated by paced papillary muscles isolated from control animals or animals expressing DAAO and treated with oral D-alanine for 4 weeks, with and without isoproterenol stimulation (100 nM). b, c Isometric contractile force and d, e relaxation times for papillary muscles at baseline and after treatment with the beta-adrenergic agonist isoproterenol (100 nM). Isoproterenol contractile force is represented as the fold change from the baseline developed tension in the absence of agonist. *p < 0.05 and **p < 0.01 by t-test. f Immunoblots of cardiac lysates from Ctrl and DAAO-infected animals and probed for phospho-phospholamban (PLN) at phospho-serine 16 and phospho-threonine 17, and for total cardiac troponin T (cTnT). g Densitometry of immunoblots shown in f. **p < 0.01 by ANOVA. Immunoblotting of the same lysates for YFP, a component of HyPer, did reveal some heterogeneity in efficiency of expression from animal to animal, but did not correlate with signaling or physiologic responses (Supplementary Figures 1A and 1B). h Relative changes in expression of alpha MHC (Myh6) and beta MHC (Myh7) in hearts from DAAO (red squares) vs. Ctrl (blue circles) infected animals. i Relative changes in expression of atrial natriuretic peptide (Nppa) in hearts of control (Ctrl) and DAAO-expressing animals treated with oral D-alanine for 4 weeks. ***p < 0.001 by t-test. j Relative expression of B-type natriuretic peptide (Nppb) in hearts from Ctrl and DAAO animals. *p < 0.05 by t-test. Direct measurement of ANP and BNP protein levels by ELISA in cardiac lysates from control (Ctrl) and DAAO-expressing animals. *p < 0.05 by t-test. Data are represented as mean ± standard error

Fig. 5

Changes in tissue and plasma cardiac biomarkers with chronic DAAO activation. a Tissue levels of atrial (ANP) and B-type (BNP) natriuretic peptide in cardiac lysates from control (Ctrl) and DAAO-expressing animals fed D-alanine for 4 weeks. *p < 0.05 by t-test. b Plasma levels of ANP and BNP in Ctrl and DAAO-expressing animals chronically fed D-alanine. *p < 0.05 by t-test. c Plasma levels of the cardiac biomarker troponin I (cTnI) in Ctrl and DAAO animals. ***p < 0.001 by t-test. Data are represented as mean ± standard error

Fig. 6

Changes in markers of oxidative stress and fibrosis after chronic activation of DAAO. a Relative changes in expression of the Nrf2 targets Hmox1, Nqo1, Sxn1, and Txnrd1 and the NF-κB targets Il1b, Tnfa, Icam1, and Nos2 in hearts from animals infected with control AAV9 (Ctrl) and DAAO virus measured by qPCR. Distributions for animals infected with control virus can be found on Supplementary Figure 4C. **p < 0.01 and ***p < 0.001 by ANOVA. b Relative changes in expression of the reductive enzymes Prdx1, Prdx2, Prdx3, Gpx1, Gpx3, Txn1, and Txn2 in Ctrl and DAAO hearts measured by qPCR. Distributions for animals infected with control virus can be found on Supplementary Figure 4D. c Reduced (GSH) measured in hearts from animals expressing DAAO vs. Ctrl. **p < 0.01 by t-test. d Total glutathione measured in hearts from DAAO-expressing vs. control animals treated with D-alanine for 4 weeks. *p < 0.05 by t-test. e Immunoblot and densitometry for the fibrotic marker galectin-3 in cardiac lysates from Ctrl and DAAO animals. f Relative changes in expression of the fibrosis-associated transcripts Col1a1, Col3a1, Tgfb1, and Mmp2 in hearts from Ctrl (blue circles) and DAAO (red squares) animals. No significant differences were observed by ANOVA. g Representative histology of hearts from Ctrl and DAAO animals stained for collagen with Masson’s trichrome stain. h Masson trichrome-stained cardiac tissue sections quantitatively analyzed for fractional area of fibrosis. The images shown are representative of n = 4 animals from each group, which were pooled for statistical analysis. Data are represented as mean ± standard error