2022, Nature Cardiovascular Research
Z. Fu1 & K. R. King Groups
Abstract
- 심근경색에서 BZ는 infact zone 과 Remote zone 을 나누는 지리적으로 복잡하고 생물학적으로도 어려운 지역
- 세포적,분자적 심근세포의 BZ는 아직 잘 알려지지 않았는데, 이는 미세절제술이 어쩔 수 없이 RZ, IZ 를 포함하고 있기 때문
- 이 논문에서는 싱글셀 기법과 공간전사체 기술을 이용하여 cardiomyocyte 전사체를 중심으로 하여 BZ 를 BZ1, BZ2 로 재정의함.
- BZ1 : Nppa+ / Xirp2- | IZ, 정상세포와 RZ구분
- BZ2 : Nppa+ / Xirp2+ | IZ에 있는 형태적으로 변형된 cardiomyocyte 를 나타내며 이는 세포골격 근섬유세포와 같은 위치에 존재하고있음.
- 기계적인 자극이 BZ 유전자를 유도하기에 충분했고,
- 이 논문에서는 ‘loss of neighbor’ 라는 가설을 통해 ischemic cell death 가 어떤 방식으로 BZ을 불안정화시키는지 설명
IZ : Region of cell death
RZ : Normal myocardium
BZ에서는 infacrt expansion, ventricular redmodeling, 부정맥 시작, CM proliferation, renereration 등이 일어나는 것으로 보고되어왔다.
Zone 은 보통 조직학적으로 CM 의 생김새에 다라 정해지는데, 초기 심근경색에서는 IZ 에서 죽어가는 CM들이 세포괴멸로 이르는 intracellular contraction band 를 보여준다. 반면 BZ 에서 살아남는 CM들은 상대적으로 정상 세포 형태를 보여주기는 하지만 이들은 RZ 와 기능적으로 구별된다.
시간이 지나며 BZ가 발달하고, 확장되면서 뇌실확장과 wall thinning, rupture 까지 일어나게 된다.
임상적으로 이러한 remodeling 은 progressive symptomatic heart failure 까지 이어지게 된다. 허혈성 세포죽음과 Remodeling 을 제한하는 치료제의 발달에도 불구하고, 허혈성 심장질환은 아직도 사망률 에서 높은 순위를 차지하고 있다.
BZ를 결정하는 것들은 많이 알려져 있지 않지만 세포적으로, 분자적으로 그 부분을 고립하는 것이 어렵기 때문이다. BZ는 보통 해부학적으로 정의되는 하지만 IZ, RZ 와 같이 있어서 쉽게 분리하기는 어렵다.
하지만 최근 싱글셀과 공간전사체 기술들이 발전하면서, 기계적으로 분리해낼 필요 없이 확인하는 것이 가능해지고 있다. 이 연구에서는 싱글셀과 공간전사체를 이용하여, 싱글셀, 공간전사체 패턴으로 BZ가 정의될 수 있다는 것을 보이고, BZ CM 이 2개로 나누어질 수 있으며 ( BZ1, BZ2 ) 각자 microenvironment 가 다르다는 것을 보인다.
BZ1은 RZ 의 경계에서 수백 mircometer 의 두께를 가지고 있으며, BZ2는 IZ 의 경계에서 수십 micrometer의 경계를 가지고 있다.
허헐성과 비허혈성 을 비교하며, 이 연구에서는 BZ CM cluster 에서 시공간적 발달을 정의하고 그 안의 mechanostranduction program 을 설명하기 위해 loss of neighbor 가설을 발전시킨다.
Result
Result
We collected myocardial tissue extending from the non-infarcted RZ to the frankly infarcted IZ to ensure com- prehensive capture of BZ biology. The resulting count matrices were integrated with our previously published scRNA-seq datasets collected at similar time points after MI to create a unified sc/snRNA-seq object with clusters representing CMs, fibroblasts, endothelial cells (ECs), smooth muscle cells (SMCs), neutrophils, dendritic cells (DCs), mono- cytes and macrophages (Extended Data Fig. 1e,f).
We began by analyzing CM transcripts since they are the cell type
sufficient for pathologists to morphologically recognize the BZ in histological sections.
Subclustering of CMs revealed a continuum of five distinct yet overlapping transcriptional states (CM1–CM5) suggestive of a regulated multistep progression
Based on cluster composition changes after
injury, gene dynamics and spatial distribution, clusters were collapsed into three major functional groups as described below (Fig. 1b, right: RZ, purple; BZ1, maroon; BZ2, red).
non-infacted, infacted sample 을 같이 clustering 하여 CM subcluster 만듬 : 대부분이 CM1에 있었고, CM2,3,4,5 는 cntrl 에 별로 없었음.
CM4,5 는 Xirp2, Flnc, Des 가 과발현되어있었는데 이는 급성 심근경색의 사망과 연관되어있는 유전자들이었고, 이들의 GO 결과 mechaniscal instability 에 대한 결과가 주로 나왔음.
CM5 는 CM4의 subtype 으로 보이는 island cluster 였는데 여기서는 hypoxia-inducible factyor 조절 유전자들이 과발현되어있었음. 이는 아마 활성화된 허혈성 CM일 가능성이 있음.
CM4,5 는 Mhrt를 포함하는 CM1유전자들이 저발현되는 양상을 띄었는데, motif enrichment 분석 결과 CM1은 Mef2 결합 부분이 많은 것으로 확인되었고 이는 최근 injury 후 CM과 Mef 가 늘어난다는 보고와 일치함.
Nppa and Ankrd1 were recently identified in infarct BZs using single-gene in situ hybridization
Ankrd1 is often considered a YAP-regulated gene involved in mechanotransduction and prolif- eration during development and cancer
Interestingly, both CM2/3
and CM4/5 were enriched for TEAD binding sites, which is the DNA binding partner involved in mediating YAP/TAZ signaling
Further, they expressed genes that encode proteins involved
in cardiac conduction including Cx43 (Gja1), Scn5a and Ryr2 that are collectively regulated by Tbx5 and Pitx2.
Additionally, they also expressed Nppa and Nppb, which encode the natriuretic peptides, stretch-activated clinical biomarkers of heart failure, suggesting that this CM population is under mechanical stress.
CM transcriptomes define spatially distinct BZ1 and BZ2
Since CM1s represented transcriptionally normal RZ myocytes and because IZ myocytes are necrotic and lack stable transcripts, we hypoth- esized that CM2/3 and CM4/5 may represent transcriptionally distinct areas of the BZ.
Spatial clustering 한 다음에 snRNA annotation을 붙힘. -> snRNA 에서의 CM DEG 의 top 들의 점수를 합쳐서 공간적으로 나뉘는 것을 확인.
This was done by first evaluating whether each spatial cluster was CM-rich (high CM gene-set score) and thus labeled either RZ or BZ, or whether it was CM-poor (low CM gene-set score) and thus labeled IZ due to presumed ischemic cell death.
As predicted by the snRNA-seq data, RZ scores were most elevated near the septum in regions
Taken together, these data show that after ischemic injury, CM transcriptomes form a continuous trajectory cor- responding to spatial position within the internal BZ, thus validating the ‘transcriptional microdissection’ method.
Non-myocytes of the BZ
MI results in the recruitment of millions of leukocytes into the IZ from hematopoietic reservoirs.
the distribution of leukocyte subsets in space and their relationship to the newly defined BZ is undefined
BZ1 and BZ2 scores were highest in IZ4 (compared to IZ1–IZ3) and Isg15 and Irf7 were most elevated in BZ2 (compared to RZ and BZ1) (Fig. 3g). This suggests that type I IFN signaling, which when inhibited leads to improved survival after MI, is mediated by or directly impacts BZ CMs
Fibroblasts in the BZ are known to transition from proinflamma-
tory to profibrotic phenotypes,
Consistent with previous reports, we identified inactivated and activated fibroblasts by the expression of gelsolin (Gsn) and actin-alpha 2, smooth muscle (Acta2) with additional underlying heterogeneity
As expected, we found inactivated fibroblasts associated with RZ CMs and BZ1 CMs (Fig. 4c,d). Proinflammatory fibroblasts (Cxcl5HI
), PostnHI and replicating fibroblasts, while homogenously distrib-
uted throughout the IZ, also colocalized with BZ2 CMs (Fig. 4c,d and Extended Data Fig. 7e).
Spatial correlation analyses sup- ported these claims (Extended Data Fig. 7c,d)
Fibroblast는 MI초기에 proinflammatory phenotype 에서 myofibroblast phenotype 으로 변화하는 것으로 알려져 있음. 논문의 연구결과는 이 과정에서 BZ1, BZ2 CM이 기여하는 바가 있음을 시사하고있는데,
These data showed that BZ2 CMs preferentially localized to PostnHI
fibroblasts compared to BZ1 CMs, in agreement with capture-
based spatial transcriptomics (Fig. 4g,h).
PostnHI fibroblasts express several genes encoding for matricellular proteins, which have been previously associated with the BZ, notably secreted protein acidic and cysteine rich (Sparc), tenascin C (Tnc), periostin (Postn) and osteopontin (Opn)
In this study, our data show that matricellular-expressing fibroblasts preferentially localize to BZ2 CMs. Taken together, these data provide a spatial transcriptomic atlas of immune and stromal subpopulations in the infarcted heart and suggest multiple distinct inflammatory, fibrotic and matricellular microenvironments surrounding BZ CMs.
Onset and evolution of the transcriptional BZ
Instead, we propose that when cells die due to ischemia, the surviving neighboring cells experience a resulting mechanical instability due to the highly asymmetric loss of load, leading to mechanotransduction changes that activate cell signaling and not only affect the immediate neighbors but also propagate surprising distances leading to the development of BZ1 transcriptional signatures hundreds of micrometers away.
Taken together, our data show that the transcriptional BZ develops rapidly, within hours of ischemia, in regions neighboring detached, frankly ischemic CMs.
The transcriptional BZ in mechanical injury models
Integration of the resulting spatial count matrices with data from ischemic models showed that the NP injury recapitulated MI-induced CM transcrip- tomes with comparable fractions of RZ, BZ1, BZ2 and IZ pixels in the excised areas and elevated BZ1 and BZ2 gene scores relative to sham (Fig. 7c–e).
… Taken together these data show that NP injury is sufficient to induce the transcriptional BZ phenotype.
We subjected mice to either a sin- gle high-dose of ISO or TAC and performed spatial transcriptomics after 72 h (Fig. 7g).
Finally, we repurposed the canonical in vitro scratch assay to test our LON hypothesis in confluent cultures of isolated neonatal rat ventricular myocytes (NRVMs) (Fig. 7j). The physical scratches in the culture plates separated myocytes from their neighbors and we measured their transcriptional responses to the mechanical separation using quantitative PCR. The loss of CM neigh- bors transcriptionally upregulated the BZ2 genes, Flnc and Xirp2, and to a lesser extent the BZ1 gene, Ankrd1, compared to unscratched Ctrl plates. Taken together, these data show that nonischemic, mechanical trauma is sufficient to induce the development of BZ transcriptional phenotypes and provides support for our LON hypothesis.
Discussion
이 논문에서는…
We found that the transcriptional BZ develops rapidly, within
hours after ischemia, arguing for a direct response by CMs rather than an indirect response to infiltrating immune cells.
Our report is not the first to show that BZ CMs express unique tran-
scriptional fingerprints. In situ hybridization has shown that CMs selec- tively express candidate genes at the infarct BZ in mice and humans3
.
Our work confirms, extends and generalizes these observations by showing that unbiased transcriptional profiling of CM nuclei encodes spatial context within the infarcted heart. The gene sets revealed by single-nucleus transcriptomics add to the growing body of literature surrounding BZ CM subsets and transcriptional regulators such as Yap/ Tead, Pitx2 and Mef2−within the injured heart3,37,46,47,66,67 . In addition, wedefined local stromal and immune contexts within the RZ, BZ and IZ, topics that will require dedicated mechanistic exploration.
The present report raises many questions for future studies. What
are the molecular mechanisms that transmit information from the IZ to BZ1 cells hundreds of micrometers away? What are the functions and fates of BZ1 and BZ2 CMs? Do BZ1 and BZ2 have proliferative potential? Do BZ2 CMs represent a recoverable population or are they destined to die? Do BZ2 CMs represent the evolving edge of ‘infarct expansion’ and if so, is the process modifiable to reduce ‘infarct size’ and progression to heart failure? Future studies are needed to shed light on the multiscale biology and organ-level significance of BZ CM subsets and their stro- mal, immune and vascular contexts. Such studies will be challenging because the transcriptional and histological techniques employed in this work only capture snapshots in time, while the underlying pro- cesses are undoubtedly dynamic. While bioinformatics methods based on pseudotime can reveal some dynamics, techniques such as lineage tracing will be critical for defining how the transcriptional BZ evolves across space and time and how it is linked to cell fate. In conclusion, by defining the single-cell spatial transcriptomes
of the BZ specifically, and the infarcted heart more generally, we hope to offer a reference dataset for future mechanistic studies aimed at therapeutically modulating the determinants of ischemic heart disease for clinical benefit.
Methods
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