Cardiac regeneration and development

Question 1

Rawles et al 1943

Answer 1

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

Question 2

Kelly et al 2001

Answer 2

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

Question 3

Lescroart et al 2014

Answer 3

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

Question 4

Prall et al 2007

Answer 4

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

Question 5

Tyser et al 2021

Answer 5

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)

Question 6

Tyser et al 2016

Answer 6

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.

Question 7

Bogers et al 1989

Answer 7

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

Question 8

Mikawa et al 1992

Answer 8

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

Question 9

Gittenberger-de-Groot et al 2007:

Answer 9

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

Question 10

Cai et al 2008

Zhou et al 2008

Answer 10

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

Question 11

Red-Horse et al 2010

Answer 11

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

Question 12

Su et al. (2018) What determines if cells are to become arterial or venous?

Answer 12

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

Question 13

Tian et al 2014

Answer 13

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

Question 14

Phansalkar et al. eLife 2021: TWO POPULATIONS significance

Answer 14

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

Question 15

Dube et al 2017

Answer 15

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

Question 16

Räsänen et al 2021

Answer 16

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

Question 17

Payne et al 2019

Answer 17

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

Question 18

Smart et al 2011

Answer 18

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

Question 19

Viera et al 2017

Answer 19

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)

Question 20

Orlic et al 2001

Answer 20

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

Question 21

Balsam et al 2004

Answer 21

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

Question 22

Chong et al 2014

Answer 22

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)

Question 23

Funakoshi et al 2021

Answer 23

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

Question 24

Vagnozzi et al 2020

Answer 24

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

Question 25

Bergmann et al 2009

Answer 25

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

Question 26

Endogenous regeneration

Answer 26

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.

Question 27

Puente et al 2014

Answer 27

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)

Question 28

Cardoso et al 2020

Answer 28

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

Question 29

Bae et al 2021

Answer 29

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

Question 30

Zhao et al 2019

Answer 30

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

Question 31

Healen et al 2011

Answer 31

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

Question 32

Bersell et al 2009

Answer 32

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)

Question 33

Pollizzotti et al 2015

Answer 33

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.

Question 34

Faeh et al 2009

Answer 34

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!

Question 35

Bon-Mathier et al 2020

Answer 35

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)

Question 36

Gonzalez-Rosa et al 2018

Answer 36

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