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.