A remarkable adaptive paradigm of heart performance and protection emerges in response to marked cardiac-specific overexpression of ADCY8
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Evaluation Summary:
The study is overall well-planned and the amount of data presented by the authors is impressive. The work nicely incorporates animal-level physiology (echocardiography data), tests for known canonical markers of hypertrophy, and then delves into an unbiased analysis of the transcriptome and proteome of LV tissue in bulk. The techniques and analyses in the study are adequately executed and within the realm of expertise of the Lakatta laboratory. This study is a necessary and crucial first step to extensively phenotype this mouse line and generate hypotheses for further work.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TG AC8 ) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TG AC8 , and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TG AC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TG AC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TG AC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TG AC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.
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Evaluation Summary:
The study is overall well-planned and the amount of data presented by the authors is impressive. The work nicely incorporates animal-level physiology (echocardiography data), tests for known canonical markers of hypertrophy, and then delves into an unbiased analysis of the transcriptome and proteome of LV tissue in bulk. The techniques and analyses in the study are adequately executed and within the realm of expertise of the Lakatta laboratory. This study is a necessary and crucial first step to extensively phenotype this mouse line and generate hypotheses for further work.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Tarasov and colleagues provide data that extensively phenotypes TGAC8 mice, which exhibit a cAMP-mediated increase in cardiac workload prior to developing heart failure. The authors confirm data from prior studies, showing increased cardiac output mediated by changes in heart rate with similar ejection fraction. Interestingly, canonical markers of LV hypertrophy did not differ from wildtype mice at the time period studied. The LV demonstrated proliferation of small cardiomyocytes and a network of interstitial non-cardiac myocytes. Transcriptomic and proteomic analyses of bulk LV tissue in TGAC8 mice compared to wildtype found pathways involved in immune responses, ROS scavenging, proliferation, and apoptosis to be activated in TGAC8 mice. Similarly, metabolic profiles shifted from fatty acid oxidation to …
Reviewer #1 (Public Review):
Tarasov and colleagues provide data that extensively phenotypes TGAC8 mice, which exhibit a cAMP-mediated increase in cardiac workload prior to developing heart failure. The authors confirm data from prior studies, showing increased cardiac output mediated by changes in heart rate with similar ejection fraction. Interestingly, canonical markers of LV hypertrophy did not differ from wildtype mice at the time period studied. The LV demonstrated proliferation of small cardiomyocytes and a network of interstitial non-cardiac myocytes. Transcriptomic and proteomic analyses of bulk LV tissue in TGAC8 mice compared to wildtype found pathways involved in immune responses, ROS scavenging, proliferation, and apoptosis to be activated in TGAC8 mice. Similarly, metabolic profiles shifted from fatty acid oxidation to glycolysis.
The study is overall well-planned and the amount of data presented by the authors is impressive. The work nicely incorporates animal-level physiology (echocardiography data), tests for known canonical markers of hypertrophy, and then delves into an unbiased analysis of the transcriptome and proteome of LV tissue in bulk. The techniques and analyses in the study are adequately executed and within the realm of expertise of the Lakatta laboratory. This study is a necessary and crucial first step to extensively phenotype this mouse line and generate hypotheses for further work.
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Reviewer #2 (Public Review):
Tarasov et al. present an impressive amount of work in their in-depth assessment of a murine model of chronic stress in a transgenic line with constitutively active AC/cAMP/PKA/Ca2+ signaling that spans cardiac structure, function, cellular architecture, gene and protein expression, mitochondrial function, energetics and more. Exploration of multiple cellular pathways throughout the manuscript and as summarized in Figure 16 help characterize this murine model and serves as a first step in using this model to understanding the effect of chronic stress on the heart. The conclusions of the manuscript are well-supported by the data, and I have the following comments:
Strengths:
1. The authors present echocardiographic, histologic, electrocardiographic, neurohormonal quantification, protein synthesis/degradation, …
Reviewer #2 (Public Review):
Tarasov et al. present an impressive amount of work in their in-depth assessment of a murine model of chronic stress in a transgenic line with constitutively active AC/cAMP/PKA/Ca2+ signaling that spans cardiac structure, function, cellular architecture, gene and protein expression, mitochondrial function, energetics and more. Exploration of multiple cellular pathways throughout the manuscript and as summarized in Figure 16 help characterize this murine model and serves as a first step in using this model to understanding the effect of chronic stress on the heart. The conclusions of the manuscript are well-supported by the data, and I have the following comments:
Strengths:
1. The authors present echocardiographic, histologic, electrocardiographic, neurohormonal quantification, protein synthesis/degradation, mitochondrial, gene and protein expression profiling, and metabolism data in their assessment of this model.
2. The verification of increased transcripts of AC and PKA activation in this transgenic line provided validation for the model.
3. The pathway analyses for both gene and protein expression profiling help supports the authors' claim of the importance of differences noted in the various pathways between the transgenic line and controls.
4. The investigators posit that there is decreased wall stress and adequate energy production due to a shift in metabolism. These findings would suggest that this model would be suitable for that of an athlete's heart, which is characterized by thickened left ventricular walls without a compromise in function. However, the mice do develop heart failure after 1 year without a sense of mechanism despite the wealth of data provided. Are the authors able to comment on what changes described in this study of this transgenic line may be deleterious in the long run?
Weaknesses:
1. As acknowledged by the investigators, this is a hypothesis-generating rather than hypothesis-testing study.
2. The investigators posit that there is decreased wall stress and adequate energy production due to a shift in metabolism. These findings would suggest that this model would be suitable for that of an athlete's heart, which is characterized by thickened left ventricular walls without a compromise in function. However, the mice do develop heart failure after 1 year without a sense of mechanism despite the wealth of data provided. Are the authors able to comment on what changes described in this study of this transgenic line may be deleterious in the long run?
3. Figure 5B is referenced to support the claim regarding beta adrenergic receptor desensitization, but the data show catecholamine levels in tissue. I would have expected receptor expression analysis to suggest up/downregulation of receptors at the membrane to support this claim.
4. Changes in ion channel (e.g. KCNQ1 and KCNJ2) gene and protein expression were described but not validated by assessment of change in function.
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Reviewer #3 (Public Review):
Tarasov et al have undertaken a very extensive series of studies in a transgenic mouse model (cardiomyocyte-specific overexpression of adenylyl cyclase type 8) that apparently resists the chronic stress of excessive cAMP signaling for around a year or so without overt heart failure. Based on the extensive analyses, including RNAseq and proteomic screening, the authors have hunted for potential "adaptive" or "protective" pathways. There is a wealth of information in this study and the experiments appear to have been carefully performed from a technical viewpoint. Many interesting pathways are identified and there is plenty of information where additional experiments could be designed.
General comments
1. Ultimately, this is a descriptive and hypothesis-generating study rather than providing directly proven …Reviewer #3 (Public Review):
Tarasov et al have undertaken a very extensive series of studies in a transgenic mouse model (cardiomyocyte-specific overexpression of adenylyl cyclase type 8) that apparently resists the chronic stress of excessive cAMP signaling for around a year or so without overt heart failure. Based on the extensive analyses, including RNAseq and proteomic screening, the authors have hunted for potential "adaptive" or "protective" pathways. There is a wealth of information in this study and the experiments appear to have been carefully performed from a technical viewpoint. Many interesting pathways are identified and there is plenty of information where additional experiments could be designed.
General comments
1. Ultimately, this is a descriptive and hypothesis-generating study rather than providing directly proven mechanistic insights. T
-Given several prior studies reporting a detrimental effect of chronically increased cAMP signaling, what is it that is different in this model? Is it something specific about AC8? Is it to do with when (in life) the stress commences?
- Is the information herein relevant to stress adaptation in general or is it just something interesting in this specific mouse model?2. None of the pathways that are apparently activated were directly perturbed so their mechanistic role requires further study.
Specific
1. The strain of the mice and their sex needs to be stated as well as the exact age at which the various assays were performed.
2. The hearts of the Tg mice have more cardiomyocytes but which are smaller. Since there is no observed increase in proliferation of cardiomyocytes, how (or when) did this increase in cell number occur?
3. While the mice do not show an increased mortality up to 12 months of age, HR/CO/EF are poor indices of contractile function. Data on end-systolic elastance or perhaps echo-based LV strain indices which will be relatively load-independent would be useful.
4. Quite a lot of conclusions are made relating to metabolism. However, this is entirely based on gene expression or protein levels. Given the substantial role of allosteric regulation in metabolic control, as well as the interconnectedness of metabolic pathways, ultimately any robust conclusions need to be based on an assessment of activity of key pathways. -
Author Response
Reviewer #1 (Public Review):
Tarasov and colleagues provide data that extensively phenotypes TGAC8 mice, which exhibit a cAMP-mediated increase in cardiac workload prior to developing heart failure. The authors confirm data from prior studies, showing increased cardiac output mediated by changes in heart rate with similar ejection fraction.
The above is slightly incorrect as stated. Our results section stated that HR and EF were increased in TGAC8, but that stroke volume did not differ by genotype. Thus 30% increase in cardiac output in TGAC8 was attributable to the increased HR.
The study is overall well-planned and the amount of data presented by the authors is impressive. The work nicely incorporates animal-level physiology (echocardiography data), tests for known canonical markers of hypertrophy, and then delves …
Author Response
Reviewer #1 (Public Review):
Tarasov and colleagues provide data that extensively phenotypes TGAC8 mice, which exhibit a cAMP-mediated increase in cardiac workload prior to developing heart failure. The authors confirm data from prior studies, showing increased cardiac output mediated by changes in heart rate with similar ejection fraction.
The above is slightly incorrect as stated. Our results section stated that HR and EF were increased in TGAC8, but that stroke volume did not differ by genotype. Thus 30% increase in cardiac output in TGAC8 was attributable to the increased HR.
The study is overall well-planned and the amount of data presented by the authors is impressive. The work nicely incorporates animal-level physiology (echocardiography data), tests for known canonical markers of hypertrophy, and then delves into an unbiased analysis of the transcriptome and proteome of LV tissue in bulk. The techniques and analyses in the study are adequately executed and within the realm of expertise of the Lakatta laboratory. This study is a necessary and crucial first step to extensively phenotype this mouse line and generate hypotheses for further work.
Reviewer #2 (Public Review):
Tarasov et al. present an impressive amount of work in their in-depth assessment of a murine model of chronic stress in a transgenic line with constitutively active AC/cAMP/PKA/Ca2+ signaling that spans cardiac structure, function, cellular architecture, gene and protein expression, mitochondrial function, energetics and more. Exploration of multiple cellular pathways throughout the manuscript and as summarized in Figure 16 help characterize this murine model and serves as a first step in using this model to understanding the effect of chronic stress on the heart. The conclusions of the manuscript are well-supported by the data, and I have the following comments:
Strengths:
The authors present echocardiographic, histologic, electrocardiographic, neurohormonal quantification, protein synthesis/degradation, mitochondrial, gene and protein expression profiling, and metabolism data in their assessment of this model.
The verification of increased transcripts of AC and PKA activation in this transgenic line provided validation for the model.
The pathway analyses for both gene and protein expression profiling help supports the authors' claim of the importance of differences noted in the various pathways between the transgenic line and controls.
The investigators posit that there is decreased wall stress and adequate energy production due to a shift in metabolism.
As written, this statement does not exactly reflect what we had intended to communicate in the paper. We did not posit, that LV wall stress was reduced in TGAC8, but that it must be reduced compared to WT on the basis of Laplace’s Law because of a substantial reduction of LV cavity volume. We also did not posit that energy production is due to a shift in metabolism, but rather, that adaptations in energy metabolism resulted in adequate energy production to meet, what appeared to us to be a marked increase in energy demand in TGAC8 vs WT, based on our observation that transcriptome and proteome gene ontology (GO) terms that differed in TGAC8 vs WT, covered nearly all biological processes and molecular functions within nearly all compartments of the LV myocardium.
These findings would suggest that this model would be suitable for that of an athlete's heart, which is characterized by thickened left ventricular walls without a compromise in function.
Although the chronic increase in cardiac output in TGAC8 heart simulates that of an athlete’s heart during exercise, LV cavity volume at rest is larger in the endurance trained heart and this is associated with bradycardia. In these aspects, the TGAC8 heart differs from the endurance trained heart (perhaps because it does not have sufficient rest periods between bouts of exercise, as does the endurance trained heart). In the discussion section of the manuscript, we noted several features that differed between the TGAC8 vs the endurance trained heart.
However, the mice do develop heart failure after 1 year without a sense of mechanism despite the wealth of data provided. Are the authors able to comment on what changes described in this study of this transgenic line may be deleterious in the long run?
Heart failure in the long run, had first been described in the TGAC8 mouse by Mougenot et. al. (ref 10 in our manuscript) who performed numerous biochemical and biophysical measurements in TGAC8 and WT attributed the heart failure to be a manifestation of accelerated heart aging. We are in the midst of conducting a longitudinal study of cardiac structure and function in the TGAC8 vs WT as these mice age, along with additional non-biased multi-omics analyses in order to get an overview about which of adaptive pathways that are activated in TGAC8 heart at 3 months of age become faltered with advancing age and how changes in these pathways relate to the altered cardiac structure and function of the TGAC8 as age advances. Following that, we will focus on each of these pathways employing detailed mechanistic analyses. Our provisional hypothesis is that while AC8 activity will continue to be increased as age advances, its downstream signaling will begin to fail due to age-associated changes in proteostasis and in the expression of proteins, including those involved in energy metabolism.
Weaknesses:
- As acknowledged by the investigators, this is a hypothesis-generating rather than hypothesistesting study.
Yes, we used a systems approach at first, in order to “open our eyes” so that we could get an overview of numerous changes that might have occurred in the TGAC8 heart in order to generate hypotheses that could later be tested by others and by us.”
- The investigators posit that there is decreased wall stress and adequate energy production due to a shift in metabolism. These findings would suggest that this model would be suitable for that of an athlete's heart, which is characterized by thickened left ventricular walls without a compromise in function. However, the mice do develop heart failure after 1 year without a sense of mechanism despite the wealth of data provided. Are the authors able to comment on what changes described in this study of this transgenic line may be deleterious in the long run?
We have addressed these comments above in our response to your comment #4 under strengths.
- Figure 5B is referenced to support the claim regarding beta adrenergic receptor desensitization, but the data show catecholamine levels in tissue. I would have expected receptor expression analysis to suggest up/downregulation of receptors at the membrane to support this claim.
Beta adrenergic receptor desensitization can occur due to changes in molecules that inhibit signaling that are at the receptor or at the signaling downstream of the receptor in the absence of changes in receptor number. Here is how we summed this up in our manuscript: “Numerous molecules that inhibit βAR signaling, (e.g. Grk5 by 2.6 fold in RNASEQ and 30% in proteome; Dab2 by 1.14 fold in RNASEQ and 18% in proteome; and β-arrestin by 1.2 fold in RNASEQ and 14% in proteome) were upregulated in the TGAC8 vs WT LV (Table S.3, S.5 and S.9), suggesting that βAR signaling is downregulated in TGAC8 vs WT, and prior studies indicate that βAR stimulation-induced contractile and HR responses are blunted in TGAC8 vs WT.8,11… A blunted response to βAR stimulation in a prior report was linked to a smaller increase in L-type Ca2+ channel current in response to βAR stimulation in the context of increased PDE activity.13, 14 WB analyses showed that PDE3A and PDE4A expression increased by 94% and 36%, respectively in TGAC8 vs WT, whereas PDE4B and PDE4D did not differ statistically by genotype (Figure 16-supplement 1 A). In addition to mechanisms that limit cAMP signaling, the expression of endogenous PKI-inhibitor protein (PKIA), which limits signaling of downstream of PKA was increased by 93% (p<0.001) in TGAC8 vs WT (Table S.3). Protein phosphatase 1 (PP1) was increased by 50% (Figure 16-supplement 1 A). The DopamineDARPP-32 feedback on cAMP signaling pathway was enriched and also activated in TGAC8 vs WT (Figure 15), the LV and plasma levels of dopamine were increased, and DARPP-32 protein was increased in WB by 269% (Figure 16-supplement 1 A).
Thus, mechanisms that limit signaling downstream of AC-PKA signaling (βAR desensitization, increased PDEs, PKI inhibitor protein, and phosphoprotein phosphatases, and increased DARPP32, cAMP (dopamine- and cAMP-regulated phosphoprotein)) are crucial components of the cardio-protection circuit that emerge in response to chronic and marked increases in AC and PKA activities (Figure 4 C, F).”
4. Changes in ion channel (e.g. KCNQ1 and KCNJ2) gene and protein expression were described but not validated by assessment of change in function.
Reviewer #3 (Public Review):
Tarasov et al have undertaken a very extensive series of studies in a transgenic mouse model (cardiomyocyte-specific overexpression of adenylyl cyclase type 8) that apparently resists the chronic stress of excessive cAMP signaling for around a year or so without overt heart failure. Based on the extensive analyses, including RNAseq and proteomic screening, the authors have hunted for potential "adaptive" or "protective" pathways. There is a wealth of information in this study and the experiments appear to have been carefully performed from a technical viewpoint. Many interesting pathways are identified and there is plenty of information where additional experiments could be designed.
General comments
1. Ultimately, this is a descriptive and hypothesis-generating study rather than providing directly proven mechanistic insights.
As noted in response to Reviewer #2: “Yes, we used a systems approach at first, in order to “open our eyes” so that we could get an overview of numerous changes that might have occurred in the TGAC8 heart in order to generate hypotheses that could later be tested by others and by us.”
-Given several prior studies reporting a detrimental effect of chronically increased cAMP signaling, what is it that is different in this model? Is it something specific about AC8? Is it to do with when (in life) the stress commences?
We believe it is, at least in part, due to something specific about the effects of the marked increased activity of AC8 perse, because adenylyl cyclase singling impacts nearly all aspects of our current knowledge of cell biology. Thus, due to the marked increase of AC and PKA activation in the TGAC8 heart, the transcriptome and proteome gene ontology (GO) terms that differ in TGAC8 vs. WT covered nearly all biological processes and molecular functions within nearly all compartments of the TGAC8 LV myocardium.
- Is the information herein relevant to stress adaptation in general or is it just something interesting in this specific mouse model?
In our opinion, AC8 mouse model is very relevant to stress adaptation in general, but this broad view has hardly ever been realized previously in the literature, because of the reductionist nature (by necessity) of mainstream biomedical research. For example, reports on cardiac specific overexpression of AC5 and AC6 never provided broader view on these mice and were focused only on a limited number of traits i.e., arrhythmogenesis, chronic pressure overload, contraction (Am J Physiol Heart Circ Physiol. 2015 Feb 1;308(3):H240-9; Am J Physiol Heart Circ Physiol. 2010 Sep;299(3):H707-12; Clin Transl Sci. 2008 Dec;1(3):221-7; Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9986-90; Am J Physiol Heart Circ Physiol. 2013 Jul 1;305(1):H1-8).
None of the pathways that are apparently activated were directly perturbed so their mechanistic role requires further study.
We agree and have entitled a section of our discussion “Opportunities for Future Scientific Inquiry Afforded by the Present Results” to address this plainly.
Specific
- The strain of the mice and their sex needs to be stated as well as the exact age at which the various assays were performed.
All assays were performed on 3-month-old males. This information was inadvertently not directly stated in the original submission.
- The hearts of the Tg mice have more cardiomyocytes but which are smaller. Since there is no observed increase in proliferation of cardiomyocytes, how (or when) did this increase in cell number occur?
It is likely that an increase in number of cardiomyocytes may have occurred during the embryonic stage of development (8.5 dpc), when AC8 expression begins. Since submitting our manuscript we have found that the expression level of human AC8 (the type of AC8 employed in this transgenic model) increases markedly during the embryonic period when compared to endogenous AC8 and remains elevated in both the fetal and perinatal periods.
- While the mice do not show an increased mortality up to 12 months of age, HR/CO/EF are poor indices of contractile function. Data on end-systolic elastance or perhaps echo-based LV strain indices which will be relatively load-independent would be useful.
Numerous comprehensive hemodynamic measurements have been performed previously on this mouse. For example, Mougenot et. al (Ref 10 in our manuscript), based on invasive hemodynamics analysis concluded that contractile function in the TGAC8 heart was increased at both 2 and 12 months of age. But Doppler imaging of the heart in conscience mice, unmasked, myocardial dysfunction, informed by a reduction in systolic strain rate in both old TGAC8 and WT littermates. This is why they attributed the heart failure in TGAC8 at 12 months of age to be a manifestation of accelerated aging.
We agree with your comment that end-systolic elastance ought to be measured in the TGAC8 but also end-diastolic elastance, and effective arterial elastance should be measured in order to quantify diastolic function and heart energetic coupling in the TGAC8.
- Quite a lot of conclusions are made relating to metabolism. However, this is entirely based on gene expression or protein levels. Given the substantial role of allosteric regulation in metabolic control, as well as the interconnectedness of metabolic pathways, ultimately any robust conclusions need to be based on an assessment of activity of key pathways.
We concur and have described some of the types of metabolic assessments in the last section of our discussion “Opportunities for Future Scientific Inquiry Afforded by the Present Results”: “… precisely defining shifts in metabolism within the cell types that comprise the TGAC8 LV myocardium via metabolomic analyses, including fluxomics.97 It will be also important that future metabolomics studies elucidate post-translational modifications (e.g. phosphorylation, acetylation, ubiquitination and 14-3-3 binding) of specific metabolic enzymes of the TGAC8 LV, and how these modifications affect their enzymatic activity”.
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