Consequences of PDGFRα+ fibroblast reduction in adult murine hearts
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Evaluation Summary:
A murine genetic platform reducing fibroblast expression shows normal background indicators of cardiac structure and contractile function. Yet it shows a reduced functional compromise, on ischemic or hypertrophic challenge. This suggests its value for studies of the effect of fibrosis following normal or pathological change.
(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
Fibroblasts produce the majority of collagen in the heart and are thought to regulate extracellular matrix (ECM) turnover. Although fibrosis accompanies many cardiac pathologies and is generally deleterious, the role of fibroblasts in maintaining the basal ECM network and in fibrosis in vivo is poorly understood. We genetically ablated fibroblasts in mice to evaluate the impact on homeostasis of adult ECM and cardiac function after injury. Fibroblast-ablated mice demonstrated a substantive reduction in cardiac fibroblasts, but fibrillar collagen and the ECM proteome were not overtly altered when evaluated by quantitative mass spectrometry and N-terminomics. However, the distribution and quantity of collagen VI, microfibrillar collagen that forms an open network with the basement membrane, was reduced. In fibroblast-ablated mice, cardiac function was better preserved following angiotensin II/phenylephrine (AngII/PE)-induced fibrosis and myocardial infarction (MI). Analysis of cardiomyocyte function demonstrated altered sarcomere shortening and slowed calcium decline in both uninjured and AngII/PE-infused fibroblast-ablated mice. After MI, the residual resident fibroblasts responded to injury, albeit with reduced proliferation and numbers immediately after injury. These results indicate that the adult mouse heart tolerates a significant degree of fibroblast loss with a potentially beneficial impact on cardiac function after injury. The cardioprotective effect of controlled fibroblast reduction may have therapeutic value in heart disease.
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Evaluation Summary:
A murine genetic platform reducing fibroblast expression shows normal background indicators of cardiac structure and contractile function. Yet it shows a reduced functional compromise, on ischemic or hypertrophic challenge. This suggests its value for studies of the effect of fibrosis following normal or pathological change.
(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):
Fibrotic change is a widespread biological phenomenon associated with both normal development and abnormal responses, often in response to pathological circumstances. In the heart, it is associated with both pump failure and arrhythmic change. This present study presents an intriguing murine genetic platform in which such processes are reduced. This used diphtheria toxin A (DTA) on a PDGFRa-CreERT2/+ mouse line. The authors report a reduction in ventricular, atrial and septal fibroblast density. However, this was surprisingly associated with relatively normal cardiac function with relatively normal histology and heart to body weight ratio, cardiomyocyte cross-sectional area, and ejection fractions, left ventricular (LV) chamber size, systolic and diastolic blood pressure, despite reduced collagen VI but not …
Reviewer #1 (Public Review):
Fibrotic change is a widespread biological phenomenon associated with both normal development and abnormal responses, often in response to pathological circumstances. In the heart, it is associated with both pump failure and arrhythmic change. This present study presents an intriguing murine genetic platform in which such processes are reduced. This used diphtheria toxin A (DTA) on a PDGFRa-CreERT2/+ mouse line. The authors report a reduction in ventricular, atrial and septal fibroblast density. However, this was surprisingly associated with relatively normal cardiac function with relatively normal histology and heart to body weight ratio, cardiomyocyte cross-sectional area, and ejection fractions, left ventricular (LV) chamber size, systolic and diastolic blood pressure, despite reduced collagen VI but not laminin and collagen IV levels. There were only minimal extracellular matrix proteomic changes. Furthermore, left anterior descending artery ligation left relatively moderated mortalities, unaltered changes in cardiac mass, measures of left-heart failure and LV chamber size, with actually better ejection fractions in fibroblast-ablated mice. Furthermore there was a reduced pathological compromise of cardiac function following profibrotic angiotensin II/phenylephrine challenge. Fibroblast ablation here did not affect cardiac mass or lung weight, sparing diastolic and slightly reducing systolic LV chamber size. Yet WT and fibroblast-ablated mice respectively showed slight decreases and fully recovered LV ejection fractions. These findings suggests the value of this platform for studies of the effect of fibrosis following normal or pathological change.
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Reviewer #2 (Public Review):
The authors use a CRE-lox system to activate DTA and ablate PDGFRa cells in the heart, which should comprise all fibroblasts. Their ablation efficiency is not particularly high, and indeed they observe limited effects on matrix composition at steady state, with most of the detectable effects on basement membrane components. Surprisingly, the ablated cells are apparently not replenished, which is a surprising finding but not studied in depth. The analysis also extends to the effects of myocardial infarction and Ang II/phenylephrine treatment, reporting beneficial effects in both in terms of reduction in scar area and interstitial fibrosis. Again, the cellular dynamics have not been addressed in these models, leaving the reader to wonder whether the expansion of PDGFRa cells that happens in these situations …
Reviewer #2 (Public Review):
The authors use a CRE-lox system to activate DTA and ablate PDGFRa cells in the heart, which should comprise all fibroblasts. Their ablation efficiency is not particularly high, and indeed they observe limited effects on matrix composition at steady state, with most of the detectable effects on basement membrane components. Surprisingly, the ablated cells are apparently not replenished, which is a surprising finding but not studied in depth. The analysis also extends to the effects of myocardial infarction and Ang II/phenylephrine treatment, reporting beneficial effects in both in terms of reduction in scar area and interstitial fibrosis. Again, the cellular dynamics have not been addressed in these models, leaving the reader to wonder whether the expansion of PDGFRa cells that happens in these situations led to a rapid replenishment back to normal levels, which would explain why the animals did not die as often happens when scar formation is interfered with. Interestingly, in the Ang II/phenylephrine model cardiomyocites appear to become hypertrophic without activation of embryonic genes as normally observed in pathological settings. Finally, the authors report decreased contractile force generation and calcium reuptake in isolated cardiomyocites from ablated hearts, suggesting that the lack of fibroblast has non-cell autonomous effects. This makes the analysis of the potential effects on other cells in the heart, such as pericytes and mural cells, important but this is not provided.
Overall, the paper provides a wealth of novel data, but lacks some critical information to be able to interpreter them.
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Reviewer #3 (Public Review):
In this manuscript, Kuwabara and colleagues use genetic ablation to reduce the number of fibroblasts resident to the heart. At baseline, the authors observe that fibroblast numbers stay proportionally low after ablation, but with very minimal effects to the structure or composition of the extracellular matrix. Fibroblast ablation prior to myocardial infarction is shown to be beneficial to cardiac function without affecting relative abundance of scar tissue, whereas in an Ang/PE model of fibrosis collagen deposition is impaired and systolic function is preserved.
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