A new chemogenetic heart failure model: “redox à la carte”
The newly-coined term ”chemogenetics” 37refers to experimental systems in which the activity of novel recombinant proteins (receptors, enzymes, channels) in cells is dynamically regulated by the addition or removal of specific biochemicals (ligands, substrates), permitting the reversible modulation of cellular responses. Chemogenetic approaches have been used modulate intracellular redox state in mammalian cells by expressing a recombinant yeast D-amino acid oxidase (DAAO) construct that catalyzes the oxidation of D-amino acids to their corresponding α-keto acids, in the process generating equimolar H2O2 (Figure 1)11 . Since mammalian tissues principally use L-amino acids 11, 18 , the yeast D-amino acid oxidase is quiescent until D-amino acid substrate is provided. Modulation of intracellular redox balance can be reversibly manipulated by providing or withdrawing D-amino acids to cells or tissues expressing recombinant DAAO: in the presence of D-amino acids, DAAO generates H2O2 until such time as the D-amino acid substrate is removed. The exquisite selectivity with which intracellular oxidants can be manipulated using chemogenetics leads us to term this experimental approach “redox à la carte”- redox state can be dynamically and specifically modulated using a single very simple biochemical intervention. Moreover, after adding D-amino acids to cells expressing DAAO, the H2O2 that is generated by DAAO can be detected in cells that are co-transfected with the highly specific H2O2 biosensor HyPer4 . This approach permits the concurrent chemogenetic generation of H2O2 (by DAAO) and detection of H2O2 by HyPer using live cell imaging 5, 34, 36 . These chemogenetic approaches in cultured cells have provided important insights into the intracellular metabolism and diffusion of H2O2 in different subcellular compartments.
Recently, these in vitro chemogenetic approaches have been extended to the in vivo setting: a new chemogenetic model of heart failure has been developed using DAAO to generate oxidative stress in cardiac myocytes in intact animals (rats or mice). There are many methodological challenges that needed to be addressed in order to make the transition from in vitro to in vivo chemogenetic approaches in the heart. In order to efficiently express DAAO in the heart, the yeast DAAO was targeted to cardiac myocytes by infecting mice or rats via tail vein injection using a recombinant DAAO virus that was constructed in the cardiotropic adeno-associated virus isotype 9 (AAV9) under control of the cardiac-specific cTnT promoter36 (Figure 1). The DAAO was expressed as a fusion protein along with the H2O2biosensor HyPer in order to be able to simultaneously generate (DAAO) and detect (HyPer) H2O2. Robust DAAO-AAV9 expression was detected in cardiac myocytes within 3-4 weeks of virus infection– with only nominal protein expression in skeletal muscle, but in no other tissues 36 . Addition of the DAAO substrate D-alanine (but not L-alanine) to isolated cardiac myocytes led to rapid and robust H2O2production, which was detected by the H2O2 biosensor HyPer that is expressed in the DAAO-HyPer fusion construct. These findings confirm that the recombinant DAAO-AAV9 construct is efficiently targeted and expressed in cardiac myocytes and verify the chemogenetic production of H2O2 in response to D-alanine in vitro . The stage was now set to study the in vivo effects of chronic oxidative stress in the heart by activating DAAO in living animals after infecting them with DAAO-AAV9.
So how does one selectively and effectively activate a stereoselective recombinant yeast enzyme once it has been delivered by a viral vector to the heart of a mammal? In vitro , the yeast DAAO is exquisitely stereoselective: L-alanine entirely fails to activate recombinant DAAO, while D-alanine markedly activates the enzyme36 . From this observation, one might infer that amino acid transporters might also be strictly stereoselective- and this stereoselectivity would undermine the tractability of in vivoapproaches in which the D-amino acid substrate is provided to the DAAO-infected animal parenterally or enterally. Fortunately (or fortuitously), most of the mammalian metabolite transporters responsible for the uptake of amino acids (encoded by the SLC solute carrier gene family) are not very stereoselective 30 . When mice or rats infected with DAAO-AAV9 are provided with drinking water supplemented with D-alanine, the animals drink the water avidly, and soon develop a dilated cardiomyopathy. Within 3 weeks of D-alanine feeding, the animals developed a striking reduction in ejection fraction and global longitudinal strain, accompanied by a significant enlargement of the left ventricular chamber and increased heart weight36 (Figure 2). As has been found in human heart failure, there was a marked increase in the abundance of transcripts encoding atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and cardiac beta myosin heavy chain (β-MHC), accompanied by a decrease in cardiac alpha-myosin heavy chain (α-MHC) transcript levels10, 17, 34, 36 . Moreover, markers of RNA oxidation were increased. Despite all these markers of cardiac dysfunction, longer-term exposure to D-alanine did not yield a further decline in ventricular function. It seems plausible that chronic exposure to oxidative stress elicits a broad range of protective compensatory responses in the heart, reflected by an increase in transcripts encoding “antioxidant” enzymes and by an increase in intracellular glutathione levels 36 . Despite marked ventricular dysfunction, there was no evidence of interstitial cardiac fibrosis even when the dilated cardiomyopathy phenotype was at its most extreme36 , suggesting that the cardiac dysfunction in this model is independent from fibrotic processes.
In order for chemogenetic heart failure to be a useful model for drug screening and target validation, the phenotype needs to be reversible. The striking dilated cardiomyopathy seen in animals infected with DAAO-AAV9 and fed D-alanine was found to fully resolve when the animals were treated with the angiotensin receptor blocker valsartan– administered either alone or with the neprilysin inhibitor sacubitril (Figure 2). This salutary response to drug treatment was seen even in the face of ongoing oxidative stress. Longer-term exposure to oxidative stress did eventually lead to the development of cardiac fibrosis, which was seen even when the heart failure phenotype had fully resolved with drug treatment. These observations lead to the important conclusion:significant cardiac dysfunction and ventricular remodeling may develop in the absence of cardiac fibrosis, while interstitial cardiac fibrosis may persist even after contractile dysfunction and adverse remodeling have resolved . These findings suggest that cardiac fibrosis cannot necessarily be used as a surrogate marker for cardiac dysfunction, nor does cardiac dysfunction necessarily involve cardiac fibrosis.