Cardiac regeneration and development Flashcards
Rawles et al 1943
Grafts produced by dividing avian blastoderm into small pieces then transplanting into host embryos
Examined for occurrence of cardiac muscle
Grafts taken from Anterio-lateral to the streak lead to production of cardiac crescent cells committed to cardiac fate
BUT only shows potential of cells, not necessarily what would take place in-vivo - how many populations not clear
Kelly et al 2001
nlacZ expression under FGF10 transgene - pharyngeal mesoderm specific. FGF10 not expressed in the heart naturally
nlacZ short term genetic lineage label – gives rise to LacZ protein which is very stable – undergoes spontaneous recombination to express B-galactodase – visualised by histochemistry
lacZ present in arterial pole of the heart (despite no lacZ transcripts/ FGF10 expression) due to long life of protein can conclude that cells originated from the pharangeal arches (SHF) where previously expressed
Only gives snapshot of development – need to gain temporal insight
Lescroart et al 2014
Found two temporally distinct pools of Mesp1 cells (primitive marker)
Generated a transgenic mouse, in which the doxycycline (Dox)-dependent transactivator (Mesp1–rtTA) is expressed under Mesp1 promoter – express tdTomato when Dox administrated
Upon low level doxycycline exposure at developmental increments, graded migration was observed from two distinct regions
E6.25 – FHF, E7.25 – SHF
Compelling evidence to suggest two distinct fields of cells FHF emerge from the streak first (laterally) while the SHR stays medial
However, should determination of cell destination based on follow up expression analysis of cells – snapshot of cell expression and does not follow up cells past this
Prall et al 2007
Nkx2.5 inhibits bone morphogenic protein (BMP) signaling by inhibiting Smad1, which holds the second lineage myocardial cells in a proliferative state as the first heart field/lineage differentiates
Nkx2.5 KO mouse
Qrt-PCR looking at transcripts of signalling molecules Reduced Smad1
HOWEVER Transcripts =/= equal protein expression (translation dependent)
OFT does not develop properly in KO as SHF not held in proliferative state
Tyser et al 2021
Demonstrates how these techniques has facilitated the discovery of new populations.
The Me5 cluster (antero-proximal region) was noted to be transcriptionally distinct from currently regarded populations, expressing some FHF markers (Hand1 and Tbx5), but significantly lower levels of the cardiac progenitor marker Nkx2-5.
scRNA-seq identified Mab21l2 as a unique marker of these cells
Though as RNA sequencing offers only a snapshot of transcriptional state, caution must be used when make inferences using “marker” genes to deduce lineage.
The use of a Cre driver line, expressed under Nkx2-5-Cre, overcomes this limitation by offering greater insight into lineage, revealing via lack of GFP staining that cells in the Me5 population never expressed Nkx2-5.
The distinct population was termed the Juxta cardiac field (JCF).
To investigate contribution to myocardium
Mab21l2-iCreERT2 transgenic line produced, limiting dose of tamoxifen to visualise JCF cells with YFP at specific points
Mab21I2 expression in JCF then expressed shortly after in myocardium and proepicardium (fate of cells)
Such a small dose, although minimising the marking of other cells (myocardium), meant that very few cells were marked and traced (~5 per embryo).
BUT too large of a dose = high tamoxifen conc which means may mark myocardium more quickly (see less discrete activity)
Tyser et al 2016
High resolution in-vivo calcium imaging first beats occur much earlier than previously thought - along the transition from the cardiac crescent to first heart field.
Gain synchrony and increase in frequency as blockade of NCX1 (reducing gradient for Ca2+ entry) prevented early calcium flows ECC likely mechanism
NCX inhibition was also shown to inhibit cardiomyocyte differentiation, suggesting these early heartbeats contribute to the growth and morphology of the developing heart, perhaps by activating effective calcium dependent signalling.
Bogers et al 1989
15 quail embryos were investigated using a monoclonal anti-endothelium antibody
Capillary plexus discovered, suggesting that the plexus grows into the aorta
Led to questioning of coronary network arising from the aorta
Mikawa et al 1992
Tracing study with retroviral tagging
Virus expressing B-galactosidase ovo-injection and (over 2000) eggs incubated
One day after hatching chicks killed and fixed
Labelling of coronary vasculature only occurred when embryos injected at day 17 or later and occurred most frequently when injected near dorsal mesocardium (suggesting origin here)
However, given what we know about post-injury - Does coronary vasculature development get affected by death of the chick, improve by looking in-vivo
Gittenberger-de-Groot et al 2007:
Allowed normal developmental timing of proepicardial ingrowth
Outgrowth of the PEO was inhibited by mechanical blocking with an egg shell membrane. Observed failure of coronary development (single artery) and embryonic lethality
Recused phenotype by implanting quail PEO next to blocked host chicken PEO
Cai et al 2008
Zhou et al 2008
Tbx18 projenitors (PEO) visualized with nlacZ under Tbx18 locus
Cells ended up as neither myocytes nor endothelial cells – instead fibroblasts and support cells (pericytes)
Tbx18 not expressed in Nkx2.5+ cells (myocyte) or pecam1+ cells (endothelium)
Purified cardiac endothelial cell fractions did not contain Tbx18-lineage-positive cells
Zhou found same for Wt1+ cells PEO
Red-Horse et al 2010
Use pelin-nlacZ knock-in mouse strain that selectively expresses nuclear β-galactosidase in coronary endothelial cells but not in endocardium
Source of plexus cells (which populated CV) was sinus venosus
Coronary endothelial clones were consistently obtained after induction of VE-cadherin-CreER – arrise from VE-cadherin+ endothelial cells
Supports SV model - no VE-cadherin+ cells other than sinus venosus sprouts and liver plexus are associated with the proepicardial organ
Only highlights sprouting present – limited in ability of study to give insight into outgrow path
Su et al. (2018) What determines if cells are to become arterial or venous?
RNA seq study given insight into this, statistically categorises subpopulations in scRNA data sets as continuous or discrete
Gradual conversion of vein to artery before transcriptional threshold crossed
Artery markers matched with pre-artery markers. Limited in that gene expression is often importantly guided by blood flow – pre artery and artery may have very different expression patterns
RNA seq only offers a snapshot – categorizing cells based on transient findings limited
Instead Cx40CreER RosatdTomato embryo lineage tracing of cx40+ pre artery cells later found in arteries
Cre ER induced COUP-TF2 expression before pre-artery specification blocks cells from contributing to coronary arteries, suggesting a failure to acquire pre-artery fate.
Vein-specifying transcription factor COUP-TF2 prevented plexus cells from overcoming the pre-artery threshold
Compare wt to COUP-TF2 overexpression, venous /arterial (hypothesised notch) genes not affected instead - cell cycle genes affected, more time in cell cycle stages in OE - reduction in proliferation of pre-artery is effect of COUP-TF2
Limitation: what is the stimulus needed to repress COUP-TF2 expression in certain cells? If means of COUP-TF2 control is unknown – how do we know COUP-TF2 OE accurate/representative of natural developmental expression – especially given the importance of gradient expression earlier
Test at different levels of OE/ repression
Although capacity to produce arterioles – this could potentially not occur commonly – maybe just to produce veins – under some unknown circumstance (perhaps compensation) changes
Tian et al 2014
Cre ER label vascular endothelial cells -progressive myocardial compaction was accompanied by accumulation of unlabeled coronary vessels in the inner half of the myocardial wall
pecam+ cells - Nfatc1-CreER;Rosa26RFP – endocardial cells contribute to these new arteries
COL3A1, an extracellular matrix protein, is part of the trabecular basement membrane - trapped endothelial cells (remote from the basement/ not associated with COL3A1) - capillary-like cells
Endothelial cells facing the ventricular lumen and residing on the basement membrane (contact COL3A1) retained sheetlike morphology - endocardial identit
Phansalkar et al. eLife 2021: TWO POPULATIONS significance
Single cell RNA sequencing of capillary cells from both origins
Early in development there were still clear differences between the two origin cells, but by day 17 the differences had almost disappeared.
The transcriptome only varied based on hypoxia level and position in the heart.
By adulthood there was rarely any difference even if there was hypoxia differences.
Impact of hypoxia likely declines in adulthood – perhaps an adaptation for development
Dube et al 2017
Some evidence of coronary sinus cells in adults sprouting after injury
Lineage tracing needed to prove that Apelin+ PECAM+ cells post MI - BUT ALTHOUGH, in embryo CS use apelin/APJ, these aren’t specific in adult, once sinus differentiates becomes quite heterogenous (widely expressed through the capillaries)
Need something specific for the coronary sinus. Better markers needed scRNAseq
Also some anatomical evidence of hypertrabeculation of tissue post MI
Räsänen et al 2021
Transgenic VEGF-B overexpression in myocardium in development and adulthood: enhanced endocardial-derived vessels.
Adeno-associated viral vector (AAV)–mediated VEGF-B delivery after ligation of the left anterior descending coronary artery promoted endocardium-derived vessel development into the myocardium and improved cardiac tissue remodeling and cardiac function (smaller scars histology)
Single cell RNA sequencing - Cxcl12, which binds to CXCR4, was upregulated in several clusters only in the infarcted VEGF-B–transduced heart - arterial reassembly and collateralization
BUT risks
Payne et al 2019
Post MI VEGFA-MEF2 pathway (upregulated in development and post-MI in neonate) are not active
Enhancer:reporter constructs provide an easily detected read-out – of regulatory pathway - more insight than simply looking at the expression pattern of one element (epigenetics can influence)
HLX-3 enhancer expression (under lacZ) - specifically absent in the infarct and border zone in all hearts examined at all time points
Smart et al 2011
TB4‐primed EPDCs give rise to cardiac progenitors in the injured adult heart
Early cardiogenic TFs are expressed, many cells expressing large amounts of TF
By day 14 complex mature cardiomyocytes
Activated adult EPDCs contribute SOME functional de novo cardiomyocytes to the ischaemic heart. Calcium transients match with native myocardium
Viera et al 2017
Epigenetic mechanism TB4 interacts with BRG1, essential ATPase subunit of SWI/SNF chromatin‐remodelling complex
TB4 ‐ BRG1 recruited to regulatory elements in the Wt1 locus and to other fetal genes (epicardial and cardiac progenitors)
Orlic et al 2001
Lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein by FACS on the basis of c-kit expression.
Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct.
Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells.
However does not prove it is the contribution of these cells - newly formed myocardium could arise from another source/mechanism – would need to lineage trace the c-kit cells
Balsam et al 2004
Completed such a lineage trace over time
Abundant GFP+ cells were detected in the myocardium after 10 days, but by 30 days, few cells were detectable.
GFP+ cells did not express cardiac tissue-specific markers, but rather, most of them expressed the haematopoietic marker CD45
Chong et al 2014
Human stem cells grafted into pig tail macaque post MI. Vessels grow into graft, but animals had higher risks of arrhythmia.
Accelerated idioventricular rhythm (AIVR) Non‐sustained VT (NSVT)
Funakoshi et al 2021
Transient activation of PPARa signalling and hormonal and fatty acid (FA) stimuli drives series of maturation steps in hESCs
Includes a switch from glycolysis to fatty acid oxidation (FAO)
Change metabolic pathway - produces maturation phenotype
Rod shape cells developed BUT lack of integration and lack of paracrine effects
Balance between maturity of cells (offering better structural and functional integration) but as cells develop lose their growth promoting paracrine effects (stem cell quality)
Caveat is that the metabolic profile in disease heart cells changes (heart disease commonly associated with MI) becomes inflexible over different energy profiles - additional cells may not contribute functionally with such different profiles
Vagnozzi et al 2020
Intracardiac injection of two distinct types of adult stem cell
Lead to the regional accumulation of ccr2+ macrophages post I-R injury - correlated with improved vascular performance
Causation inferred as ablated in ccr2+ KO
Does cardiac procedure itself produce this inflammatory response? – sham control did not improve in performance
Bergmann et al 2009
Carbon14 tracing post nucelar weapons testing
1% turnover of heart muscle at age 20, then by 0.45% per year at age 75. Age 50 ~45% of heart muscle has been replaced
Endogenous regeneration
Genetic image tracing experiments
Tamoxifen inducible GFP reported cardiomyocytes, then deplete tamoxifen, then complete injury, if the cardiomyocytes are replaced by existing cardiomyocytes (they inherit existing GFP label) hence no dilution of GFP would indicate this
If they were to come from stem cells, they would only later express the GFP, not enough tamoxifen to induce this so the GFP would be diluted.
Puente et al 2014
Mitochondrial maturation has been suggested as a mediator of cardiomyocyte cell cycle arrest
rt-PCR to examine mtDNA Detected ROS with indicator
Increased with age (also higher in humans than zfish)
Injection of ROS generator diquat induced accelerated cardiomyocyte cell cycle arrest.
HOWEVER apoptosis, cytokinesis assessed by sampled imaging -may not be representative of full heart – perhaps sampled tissue experienced surplus oxidative damage through being sampled
Functional measure in response to MI better (use neonatal mice)
Cardoso et al 2020
Fat deficient diet is sufficient to increase the generation of new cardiomyocytes in young mice, even though no differences were then observed after 10 weeks of age
Maintains their foetal metabolism
Highlights issue in diabetic cardiomyopathy – heart metabolism loses flexibility – influencing regenerative capacity – importantly in adults – response to therapy
Bae et al 2021
SDH inhibition via malonate, which promoted regeneration by inducing glucose metabolism
Elevated reactive oxygen species production in ischemic tissues occurs as a result of accumulation of the mitochondrial metabolite succinate during ischemia via succinate dehydrogenase (SDH)
Better than diet as likely that over long term changes to diet are counteracted by dealing with these metabolites to maintain homeostasis
BUT with co-morbidity of diabetic cardiomyopathy – reduced metabolic flexibility of the heart
Zhao et al 2019
Using a Biowire chip seeded with cell-hydrogel mixture, have constructed a platform which can generate atrial- and ventricular-specific cardiac tissue by combining the directed cell differentiation and electric field conditioning, which is very close to the simulation of distinct electric signaling in adult heart chambers.
Organoids
Healen et al 2011
Genetic deletion of the Hippo pathway component Salv in cardiac lineage gives rise to enlarged heart at birth with increased proliferation of cardiomyocytes
Investigated cardiomyocyte proliferation by double immunostaining with phosphorylated Ser10 histone H3 (pHH3) antibodies (specific to mitosis) to detect mitotic cells and sarcomeric myosin (α-MF20)
Less than 1% of control ventricular cardiomyocytes were pHH3-positive, whereas about 4.5% of Salv CKO mutant cardiomyocytes were pHH3-positive, indicating excessive cardiac proliferation in cardiomyocytes
BUT dephosphorylation occurs slowly from late anaphase to early telophase - therefore only marks cells in metaphase .. single point therefore may not mark all proliferating cells.
Moreover - upregulation of Wnt target genes
Bersell et al 2009
Injected recombinant NRG1 in 3-month-old mice, labeled with BrdU in the drinking water for 9 days, and then quantified cardiomyocyte cell-cycle activity
In control mice, we did not detect cycling cardiomyocytes in mice injected with recombinant NRG1, 14.3% ± 6.5% of mononucleated and 3% ± 1.2% of multinucleated cardiomyocytes were BrdU positive
Yet still very small percentage
Cells only in S phase (more cells or does it affect the duration of cell cycle)
Pollizzotti et al 2015
Newborn mice were subjected to cryoinjury, which induced myocardial dysfunction and scar formation and decreased cardiomyocyte cell cycle activity.
Early administration of rNRG1 to mice from birth to 34 days of age improved myocardial function and reduced the prevalence of transmural scars.
Increased ejection fraction measured with echocardiography and cMRI
TUNEL used to show significant cardio protection
In contrast, administration of rNRG1 from 4 to 34 days of age only transiently improved myocardial function
Stimulate cardiomyocyte proliferation in intact cultured myocardium from pediatric patients
Critical window for efficacy - paediactric specific therapy (beneficial as therapies for adult patients not always applicable for infant heart failure)
However less inflammation in cryoinjury than LAD ligation, although good at recapitulating scar formation, ECM deposition and inflammation reduced may impair action of NRG.
Faeh et al 2009
Switzerland - 1.4 million subjects living at different elevations a marked progressive decrease in CHD-induced mortality in people living in altitude - mortality rate induced by CHD decreased by 22% per 1000 m increased altitude
BUT chronic hypoxia!
Bon-Mathier et al 2020
Cell cultures at 3% and 20% O2 - cardiomyocytes expressed genes associated with de-differentiation (occurs prior to proliferation)
But cannot prove with the results presented here that de-differentiated cardiomyocytes undergo cytokinesis.
Live imaging needed to observe cytokinesis (AuroraB stainings)
Gonzalez-Rosa et al 2018
Enforced cardiomyocyte polyploidization has been demonstrated to reduce cardiomyocyte proliferation and to represent a barrier to heart regeneration in the zebrafish model
Ect2 expression - susceptible to polyploidization upon transient cytokinesis inhibition
Zebrafish cannot regenerate
