Synthetic torpor in the rat protects the heart from ischaemia-reperfusion injury
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eLife Assessment
This important submission from Ambler and colleagues brings new insights into how torpor conditions may confer resilience in cases of cardioprotection. It has novelty, which can be enhanced by additional in vivo support. The study is backed by solid evidence, and represents a unique interoceptive mechanism of interest.
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Abstract
During times of environmental stress, many animals enter torpor: a reversible protective physiological state typically characterised by reductions in core temperature, heart rate and oxygen consumption. Species that naturally enter this hypothermic and hypometabolic state are tolerant of ischaemia-reperfusion injury. Consequently, there is a growing interest in utilizing aspects of torpor for clinical applications, such as protection from stroke or myocardial infarction. It is currently unknown, however, whether a torpor-like state is protective in animals that do not naturally enter torpor. Using viral vector-mediated chemogenetic activation of the medial preoptic area of the hypothalamus, we induced synthetic torpor in the rat, a species that does not naturally enter torpor. We demonstrate this state is cardioprotective in an ex vivo ischaemia-reperfusion injury model with an ∼40% reduction in infarct size. Synthetic torpor-induced cardioprotection of the normothermic, isolated heart is not dependent on prior hypothermia in vivo . Phosphoproteomic analysis of cardiac tissue indicates the protective effects of synthetic torpor may be mediated by parallel activation of cell survival and stress tolerance pathways and inhibition of cell death pathways. These findings provide important insights into the mechanisms of organ protective effects of synthetic torpor states with implications for future clinical translation in humans.
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eLife Assessment
This important submission from Ambler and colleagues brings new insights into how torpor conditions may confer resilience in cases of cardioprotection. It has novelty, which can be enhanced by additional in vivo support. The study is backed by solid evidence, and represents a unique interoceptive mechanism of interest.
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Reviewer #1 (Public review):
Summary:
Torpor can be induced by chemogenetic activation of the medial preoptic area. This activation leads to protection from myocardial infarction in an isolated heart preparation despite normalization of the ambient temperature, thus, in principle, uncoupling hypothermia from torpor-induced neuroprotection. Putative pathways of protection are suggested by proteomic studies.
Strengths:
(1) Elegant strategy for inducing torpor in rats.
(2) Appropriate controls for verifying the neuron transducer.
(3) Cardiac protection is significant and appears independent of hypothermia.
(4) Interesting omic strategy to begin to find established and novel pathways mediating organ autonomous torpor-induced protection.
Weaknesses:
(1) The study would benefit from using inhibitory chemogenetics of the same neurons to …
Reviewer #1 (Public review):
Summary:
Torpor can be induced by chemogenetic activation of the medial preoptic area. This activation leads to protection from myocardial infarction in an isolated heart preparation despite normalization of the ambient temperature, thus, in principle, uncoupling hypothermia from torpor-induced neuroprotection. Putative pathways of protection are suggested by proteomic studies.
Strengths:
(1) Elegant strategy for inducing torpor in rats.
(2) Appropriate controls for verifying the neuron transducer.
(3) Cardiac protection is significant and appears independent of hypothermia.
(4) Interesting omic strategy to begin to find established and novel pathways mediating organ autonomous torpor-induced protection.
Weaknesses:
(1) The study would benefit from using inhibitory chemogenetics of the same neurons to demonstrate that this might make cardiac response to ischemia worse.
(2) Infecting an area of the brain not known to be involved in torpor would be a useful control.
(3) In vivo cardio protection seems essential as the validation of the strategy requires support that is in the intact animal.
(4) The assumption that the positive effects of torpor are mediated via a phosphoproteomic change rather than a translational or transcriptional control mechanism is not established.
(5) A 40 percent reduction in infarct size may work for genetically identical rats with no co-morbidities, but is unlikely to be significant enough to weather the variability that emerges in humans because of these differences and more. The question is not what the mechanism is, but how do we make it more robust? Overall, this is at best a preliminary data set that requires more experiments to deliver on its immense promise.
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Reviewer #2 (Public review):
Summary:
Elley and colleagues induced a synthetic torpor-like state in rats (a non-hibernating species) by chemogenetically activating neurons in the medial preoptic area of the hypothalamus. They show that this state substantially reduced cardiac infarct size in an ex vivo ischemia-reperfusion model. They further report that protection persisted when ambient temperature was raised to prevent hypothermia, and used exploratory phosphoproteomics to identify candidate cardioprotective signaling pathways.
Strengths:
This is the first demonstration that a torpor-like state is cardioprotective in a species that does not naturally enter torpor, which meaningfully advances the potential clinical utility of synthetic torpor. The experimental design is logical, and the controls are generally appropriate. The …
Reviewer #2 (Public review):
Summary:
Elley and colleagues induced a synthetic torpor-like state in rats (a non-hibernating species) by chemogenetically activating neurons in the medial preoptic area of the hypothalamus. They show that this state substantially reduced cardiac infarct size in an ex vivo ischemia-reperfusion model. They further report that protection persisted when ambient temperature was raised to prevent hypothermia, and used exploratory phosphoproteomics to identify candidate cardioprotective signaling pathways.
Strengths:
This is the first demonstration that a torpor-like state is cardioprotective in a species that does not naturally enter torpor, which meaningfully advances the potential clinical utility of synthetic torpor. The experimental design is logical, and the controls are generally appropriate. The characterisation of the responsible neuronal population using ISH against QPLOT markers adds mechanistic depth and supports the cross-species conservation argument. The phosphoproteomic analysis, though exploratory, generates plausible and biologically coherent hypotheses grounded in the hibernation literature.
Weaknesses:
The primary weakness is that the central conclusion - that hypothermia is not necessary for cardioprotection - exceeds the evidence. The thermoneutral groups were not demonstrably normothermic (36.4 vs 37.05{degree sign}C, p=0.44 with n=6), core temperature telemetry was absent in the majority of control animals contributing to the infarct endpoint, and the decisive test, i.e., a correlation between individual nadir temperature and infarct size, was never performed. Additional weaknesses include the absence of sex-stratified analysis despite known estrogenic contributions to torpor
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Reviewer #3 (Public review):
Summary:
The manuscript by Elley and colleagues describes experiments on the effects of synthetic torpor on ex vivo heart ischemia. The key aspect of the study was the use of viral-vector mediated manipulation of the hypothalamic medial preoptic area (MPA) in rats. They used AAV-CaMKIIa-hM3D(Gq). The authors report that chemogenetic activation of the MPA prior to an ex vivo heart ischemia-reperfusion insult induces cardio protection against infarct size that is independent of prior in vivo hypothermia. Phosphoproteomic analysis of cardiac tissue suggested changes in cell survival and death pathways.
Strengths:
This study has important strengths. The idea is novel. The experimental design is appropriately rationalized and fascinating. The manuscript is written and presented concisely.
Weaknesses:
The study …
Reviewer #3 (Public review):
Summary:
The manuscript by Elley and colleagues describes experiments on the effects of synthetic torpor on ex vivo heart ischemia. The key aspect of the study was the use of viral-vector mediated manipulation of the hypothalamic medial preoptic area (MPA) in rats. They used AAV-CaMKIIa-hM3D(Gq). The authors report that chemogenetic activation of the MPA prior to an ex vivo heart ischemia-reperfusion insult induces cardio protection against infarct size that is independent of prior in vivo hypothermia. Phosphoproteomic analysis of cardiac tissue suggested changes in cell survival and death pathways.
Strengths:
This study has important strengths. The idea is novel. The experimental design is appropriately rationalized and fascinating. The manuscript is written and presented concisely.
Weaknesses:
The study has important weaknesses in the experimental design and validation of the model.
(1) The study is based on the use of a DREADD-designed viral vector (AAV-CaMKIIa-hM3D(Gq) -mCherry) that is activated by 2 mg/kg IP injection of CNO. The rationale is to putatively activate the MPA. The authors show no evidence for chemogenetic activation of neurons in the MPA. This could be done using a variety of different approaches, even phosphoproteomics.
(2) The stereotaxic injections are difficult to precisely and locally place, particularly bilaterally. Figure 2F is only a schematic. It would be better to show actual low magnification brain sections (bregma +0.12 to -0.48) from a representative rat to show the placement of the AAV.
(3) The control rats were injected with AAV-CaMKIIa-EGFP. Why was EGFP used instead of mCherry for the control?
(4) Ideally, a mutant non-activatable variant of AAV-CaMKIIa-hM3D(Gq) should have been used for a better control.
(5) The authors should comment on whether there is any neurotoxicity in the MPA associated with the forced AAV expression of hM3D-Gq.
(6) Is there any inflammatory pathology seen in the MPA with AAV transduction?
(7) There are no experiments to show that the systemic torpor is specifically associated with the MPA region. Experiments should be done with injections of AAV-CaMKIIa-hM3D(Gq)-mCherry placed in other brain regions, for example, the nearby nucleus accumbens.
(8) The mapping of the distribution of neurons responsible for synthetic torpor is not mechanistic enough and is not directly to the point. While excitatory and inhibitory markers are examined, a more interesting and deeper approach would have been to use glutamate receptor antagonists to manipulate the torpor response.
(9) The ischemia and reperfusion aspects of the Lagendorff method need to be clarified. The isolated hearts are already ischemic after their removal from the rat. The reperfusion aspect is caused by reflow of blood to generate oxidative stress, but in the ex vivo model, is there really reperfusion injury?
(10) The authors show that whole animal oxygen consumption is reduced in the torpor state. The measurement is crude and most likely reflects the inactivity of the animal's skeletal muscle in the torpor state. A more relevant and direct experiment would be to do oxygen consumption (or Seahorse) assays on extracts of the isolated hearts.
(11) The authors report that the synthetic torpor induces bradycardia. There is no follow-up on this important observation. The MPA-heart connection is not analyzed. (A) Is the link through cardiovascular centers in the brainstem? (B) Is the torpor-induced bradycardia mediated through increased parasympathetic or decreased sympathetic autonomic tone? Pharmacological experiments could also be done.
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