Proliferation and Maturation: Janus and the Art of Cardiac Tissue Engineering.

During cardiac development and morphogenesis, cardiac progenitor cells differentiate into cardiomyocytes that expand in number and size to generate the fully formed heart. Much is known about the factors that regulate initial differentiation of cardiomyocytes, and there is ongoing research to identify how these fetal and immature cardiomyocytes develop into fully functioning, mature cells. Accumulating evidence indicates that maturation limits proliferation and conversely proliferation occurs rarely in cardiomyocytes of the adult myocardium. We term this oppositional interplay the proliferation-maturation dichotomy. Here we review the factors that are involved in this interplay and discuss how a better understanding of the proliferation-maturation dichotomy could advance the utility of human induced pluripotent stem cell-derived cardiomyocytes for modeling in 3-dimensional engineered cardiac tissues to obtain truly adult-level function.

Molecular basis of physiological heart growth: fundamental concepts and new players.

The heart hypertrophies in response to developmental signals as well as increased workload. Although adult-onset hypertrophy can ultimately lead to disease, cardiac hypertrophy is not necessarily maladaptive and can even be beneficial. Progress has been made in our understanding of the structural and molecular characteristics of physiological cardiac hypertrophy, as well as of the endocrine effectors and associated signalling pathways that regulate it. Physiological hypertrophy is initiated by finite signals, which include growth hormones (such as thyroid hormone, insulin, insulin-like growth factor 1 and vascular endothelial growth factor) and mechanical forces that converge on a limited number of intracellular signalling pathways (such as PI3K, AKT, AMP-activated protein kinase and mTOR) to affect gene transcription, protein translation and metabolism. Harnessing adaptive signalling mediators to reinvigorate the diseased heart could have important medical ramifications.

YTHDF1 is pivotal for maintenance of cardiac homeostasis.

The YTH-domain family (YTHDF) of RNA binding proteins can control gene expression at the post-transcriptional level by regulating mRNAs with N6-methyladenosine (m6A) modifications. Despite the established importance of m6A in the heart, the cardiac role of specific m6A-binding proteins remains unclear. Here, we characterized the function of YTHDF1 in cardiomyocytes using a newly generated cardiac-restricted mouse model. Deletion of YTHDF1 in adult cardiomyocytes led to hypertrophy, fibrosis, and dysfunction. Using mass spectrometry, we identified the necessity of YTHDF1 for the expression of cardiomyocyte membrane raft proteins. Specifically, YTHDF1 bound to m6A-modified Caveolin 1 (Cav1) mRNA and favored its translation. We further demonstrated that YTHDF1 regulates downstream ERK signaling. Altogether, our findings highlight a novel role for YTHDF1 as a post-transcriptional regulator of caveolar proteins which is necessary for the maintenance of cardiac function.

Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis.

RATIONALE: The initial hypertrophy response to cardiac pressure overload is considered compensatory, but with sustained stress, it eventually leads to heart failure. Recently, a role for recruited macrophages in determining the transition from compensated to decompensated hypertrophy has been established. However, whether cardiac resident immune cells influence the early phase of hypertrophy development has not been established. OBJECTIVE: To assess the role of cardiac immune cells in the early hypertrophy response to cardiac pressure overload induced by transverse aortic constriction (TAC). METHODS AND RESULTS: We performed cytometry by time-of-flight to determine the identity and abundance of immune cells in the heart at 1 and 4 weeks after TAC. We observed a substantial increase in cardiac macrophages 1 week after TAC. We then conducted Cite-Seq single-cell RNA sequencing of cardiac immune cells isolated from 4 sham and 6 TAC hearts. We identified 12 clusters of monocytes and macrophages, categorized as either resident or recruited macrophages, that showed remarkable changes in their abundance between sham and TAC conditions. To determine the role of cardiac resident macrophages early in the response to a hypertrophic stimulus, we used a blocking antibody against macrophage colony-stimulating factor 1 receptor (CD115). As blocking CD115 initially depletes all macrophages, we allowed the replenishment of recruited macrophages by monocytes before performing TAC. This preferential depletion of resident macrophages resulted in enhanced fibrosis and a blunted angiogenesis response to TAC. Macrophage depletion in CCR2 (C-C chemokine receptor type 2) knockout mice showed that aggravated fibrosis was primarily caused by the recruitment of monocyte-derived macrophages. Finally, 6 weeks after TAC these early events lead to depressed cardiac function and enhanced fibrosis, despite complete restoration of cardiac immune cells. CONCLUSIONS: Cardiac resident macrophages are a heterogeneous population of immune cells with key roles in stimulating angiogenesis and inhibiting fibrosis in response to cardiac pressure overload.

The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability.

Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.

Abcg2-expressing side population cells contribute to cardiomyocyte renewal through fusion.

The adult mammalian heart has a limited regenerative capacity. Therefore, identification of endogenous cells and mechanisms that contribute to cardiac regeneration is essential for the development of targeted therapies. The side population (SP) phenotype has been used to enrich for stem cells throughout the body; however, SP cells isolated from the heart have been studied exclusively in cell culture or after transplantation, limiting our understanding of their function in vivo. We generated a new Abcg2-driven lineage-tracing mouse model with efficient labeling of SP cells. Labeled SP cells give rise to terminally differentiated cells in bone marrow and intestines. In the heart, labeled SP cells give rise to lineage-traced cardiomyocytes under homeostatic conditions with an increase in this contribution following cardiac injury. Instead of differentiating into cardiomyocytes like proposed cardiac progenitor cells, cardiac SP cells fuse with preexisting cardiomyocytes to stimulate cardiomyocyte cell cycle reentry. Our study is the first to show that fusion between cardiomyocytes and non-cardiomyocytes, identified by the SP phenotype, contribute to endogenous cardiac regeneration by triggering cardiomyocyte cell cycle reentry in the adult mammalian heart.

The N6-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy.

BACKGROUND: N6-Methyladenosine (m6A) methylation is the most prevalent internal posttranscriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known. METHODS: To determine the role of m6A methylation in the heart, we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase methyltransferase-like 3 (METTL3) both in culture and in vivo. We generated cardiac-restricted gain- and loss-of-function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function. RESULTS: We measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, whereas increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for the maintenance of cardiac homeostasis. CONCLUSIONS: Our study identified METTL3-mediated methylation of mRNA on N6-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy, whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.

c-kit+ cells minimally contribute cardiomyocytes to the heart.

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit(+) cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit(+) cells amply generated cardiac endothelial cells. Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

The Role of TGF-ß Signaling in Cardiomyocyte Proliferation.

PURPOSE OF REVIEW: The loss of contractile function after heart injury remains one of the major healthcare issues of our time. One strategy to deal with this problem would be to increase the number of cardiomyocytes to enhance cardiac function. In the last couple of years, reactivation of cardiomyocyte proliferation has repeatedly demonstrated to aid in functional recovery after cardiac injury. RECENT FINDINGS: The Tgf-ß superfamily plays key roles during development of the heart and populating the embryonic heart with cardiomyocytes. In this review, we discuss the role of Tgf-ß signaling in regulating cardiomyocyte proliferation during development and in the setting of cardiac regeneration. Although various pathways to induce cardiomyocyte proliferation have been established, the extent to which cardiomyocyte proliferation requires or involves activation of the Tgf-ß superfamily is not entirely clear. More research is needed to better understand cross-talk between pathways that regulate cardiomyocyte proliferation.

Adhesive thermosensitive gels for local delivery of viral vectors.

Local delivery of viral vectors can enhance the efficacy of therapies by selectively affecting necessary tissues and reducing the required vector dose. Pluronic F127 is a thermosensitive polymer that undergoes a solution-gelation (sol-gel) transition as temperature increases and can deliver vectors without damaging them. While pluronics can be spread over large areas, such as the surface of an organ, before gelation, they lack sufficient adhesivity to remain attached to some tissues, such as the surface of the heart or mucosal surfaces. Here, we utilized blends of pluronic F127 and polycarbophil (PCB), a mucoadhesive agent, to provide the necessary adhesivity for local delivery of viral vectors to the cardiac muscle. The effects of PCB concentration on adhesive properties, sol-gel temperature transition and cytocompatibility were evaluated. Rheological studies showed that PCB decreased the sol-gel transition temperature at concentrations >1% and increased the adhesive properties of the gel. Furthermore, these gels were able to deliver viral vectors and transduce cells in vitro and in vivo in a neonatal mouse apical resection model. These gels could be a useful platform for delivering viral vectors over the surface of organs where increased adhesivity is required.

Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

BACKGROUND: Although cardiac c-kit+ cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit+ cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit+ cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit+ cells. METHODS: We used single-cell sequencing and genetic lineage tracing of c-kit+ cells to determine whether various pathological stimuli would result in different fates of c-kit+ cells. RESULTS: Single-cell sequencing of cardiac CD45-c-kit+ cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit+ cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit+ cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. CONCLUSIONS: These results demonstrate that different pathological stimuli induce different cell fates of c-kit+ cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit+ cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit+ cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit+ cells.

DUSP8 Regulates Cardiac Ventricular Remodeling by Altering ERK1/2 Signaling.

RATIONALE: Mitogen-activated protein kinase (MAPK) signaling regulates the growth response of the adult myocardium in response to increased cardiac workload or pathological insults. The dual-specificity phosphatases (DUSPs) are critical effectors, which dephosphorylate the MAPKs to control the basal tone, amplitude, and duration of MAPK signaling. OBJECTIVE: To examine DUSP8 as a regulator of MAPK signaling in the heart and its impact on ventricular and cardiac myocyte growth dynamics. METHODS AND RESULTS: Dusp8 gene-deleted mice and transgenic mice with inducible expression of DUSP8 in the heart were used here to investigate how this MAPK-phosphatase might regulate intracellular signaling and cardiac growth dynamics in vivo. Dusp8 gene-deleted mice were mildly hypercontractile at baseline with a cardiac phenotype of concentric ventricular remodeling, which protected them from progressing towards heart failure in 2 surgery-induced disease models. Cardiac-specific overexpression of DUSP8 produced spontaneous eccentric remodeling and ventricular dilation with heart failure. At the cellular level, adult cardiac myocytes from Dusp8 gene-deleted mice were thicker and shorter, whereas DUSP8 overexpression promoted cardiac myocyte lengthening with a loss of thickness. Mechanistically, activation of extracellular signal-regulated kinases 1/2 were selectively increased in Dusp8 gene-deleted hearts at baseline and following acute pathological stress stimulation, whereas p38 MAPK and c-Jun N-terminal kinases were mostly unaffected. CONCLUSIONS: These results indicate that DUSP8 controls basal and acute stress-induced extracellular signal-regulated kinases 1/2 signaling in adult cardiac myocytes that then alters the length-width growth dynamics of individual cardiac myocytes, which further alters contractility, ventricular remodeling, and disease susceptibility.

STIM1 elevation in the heart results in aberrant Ca²âº handling and cardiomyopathy.

Stromal interaction molecule 1 (STIM1) is a Ca(2+) sensor that partners with Orai1 to elicit Ca(2+) entry in response to endoplasmic reticulum (ER) Ca(2+) store depletion. While store-operated Ca(2+) entry (SOCE) is important for maintaining ER Ca(2+) homeostasis in non-excitable cells, it is unclear what role it plays in the heart, although STIM1 is expressed in the heart and upregulated during disease. Here we analyzed transgenic mice with STIM1 overexpression in the heart to model the known increase of this protein in response to disease. As expected, STIM1 transgenic myocytes showed enhanced Ca(2+) entry following store depletion and partial co-localization with the type 2 ryanodine receptor (RyR2) within the sarcoplasmic reticulum (SR), as well as enrichment around the sarcolemma. STIM1 transgenic mice exhibited sudden cardiac death as early as 6weeks of age, while mice surviving past 12weeks of age developed heart failure with hypertrophy, induction of the fetal gene program, histopathology and mitochondrial structural alterations, loss of ventricular functional performance and pulmonary edema. Younger, pre-symptomatic STIM1 transgenic mice exhibited enhanced pathology following pressure overload stimulation or neurohumoral agonist infusion, compared to controls. Mechanistically, cardiac myocytes isolated from STIM1 transgenic mice displayed spontaneous Ca(2+) transients that were prevented by the SOCE blocker SKF-96365, increased L-type Ca(2+) channel (LTCC) current, and enhanced Ca(2+) spark frequency. Moreover, adult cardiac myocytes from STIM1 transgenic mice showed both increased diastolic Ca(2+) and maximal transient amplitude but no increase in total SR Ca(2+) load. Associated with this enhanced Ca(2+) profile was an increase in cardiac nuclear factor of activated T-cells (NFAT) and Ca(2+)/calmodulin-dependent kinase II (CaMKII) activity. We conclude that STIM1 has an unexpected function in the heart where it alters communication between the sarcolemma and SR resulting in greater Ca(2+) flux and a leaky SR compartment.

Unrestrained p38 MAPK activation in Dusp1/4 double-null mice induces cardiomyopathy.

RATIONALE: Mitogen-activated protein kinases (MAPKs) are activated in the heart by disease-inducing and stress-inducing stimuli, where they participate in hypertrophy, remodeling, contractility, and heart failure. A family of dual-specificity phosphatases (DUSPs) directly inactivates each of the MAPK terminal effectors, potentially serving a cardioprotective role. OBJECTIVE: To determine the role of DUSP1 and DUSP4 in regulating p38 MAPK function in the heart and the effect on disease. METHODS AND RESULTS: Here, we generated mice and mouse embryonic fibroblasts lacking both Dusp1 and Dusp4 genes. Although single nulls showed no molecular effects, combined disruption of Dusp1/4 promoted unrestrained p38 MAPK activity in both mouse embryonic fibroblasts and the heart, with no change in the phosphorylation of c-Jun N-terminal kinases or extracellular signal-regulated kinases at baseline or with stress stimulation. Single disruption of either Dusp1 or Dusp4 did not result in cardiac pathology, although Dusp1/4 double-null mice exhibited cardiomyopathy and increased mortality with aging. Pharmacological inhibition of p38 MAPK with SB731445 ameliorated cardiomyopathy in Dusp1/4 double-null mice, indicating that DUSP1/4 function primarily through p38 MAPK in affecting disease. At the cellular level, unrestrained p38 MAPK activity diminished cardiac contractility and Ca2+ handling, which was acutely reversed with a p38 inhibitory compound. Poor function in Dusp1/4 double-null mice also was partially rescued by phospholamban deletion. CONCLUSIONS: Our data demonstrate that Dusp1 and Dusp4 are cardioprotective genes that play a critical role in the heart by dampening p38 MAPK signaling that would otherwise reduce contractility and induce cardiomyopathy.

Serine 105 phosphorylation of transcription factor GATA4 is necessary for stress-induced cardiac hypertrophy in vivo.

Cardiac hypertrophy is an adaptive growth process that occurs in response to stress stimulation or injury wherein multiple signal transduction pathways are induced, culminating in transcription factor activation and the reprogramming of gene expression. GATA4 is a critical transcription factor in the heart that is known to induce/regulate the hypertrophic program, in part, by receiving signals from MAPKs. Here we generated knock-in mice in which a known MAPK phosphorylation site at serine 105 (S105) in Gata4 that augments activity was mutated to alanine. Homozygous Gata4-S105A mutant mice were viable as adults, although they showed a compromised stress response of the myocardium. For example, cardiac hypertrophy in response to phenylephrine agonist infusion for 2 wk was largely blunted in Gata4-S105A mice, as was the hypertrophic response to pressure overload at 1 and 2 wk of applied stimulation. Gata4-S105A mice were also more susceptible to heart failure and cardiac dilation after 2 wk of pressure overload. With respect to the upstream pathway, hearts from Gata4-S105A mice did not efficiently hypertrophy following direct ERK1/2 activation using an activated MEK1 transgene in vivo. Mechanistically, GATA4 mutant protein from these hearts failed to show enhanced DNA binding in response to hypertrophic stimulation. Moreover, hearts from Gata4-S105A mice had significant changes in the expression of hypertrophy-inducible, fetal, and remodeling-related genes.

Differential expression of embryonic epicardial progenitor markers and localization of cardiac fibrosis in adult ischemic injury and hypertensive heart disease.

During embryonic heart development, the transcription factors Tcf21, Wt1, and Tbx18 regulate activation and differentiation of epicardium-derived cells, including fibroblast lineages. Expression of these epicardial progenitor factors and localization of cardiac fibrosis were examined in mouse models of cardiovascular disease and in human diseased hearts. Following ischemic injury in mice, epicardial fibrosis is apparent in the thickened layer of subepicardial cells that express Wt1, Tbx18, and Tcf21. Perivascular fibrosis with predominant expression of Tcf21, but not Wt1 or Tbx18, occurs in mouse models of pressure overload or hypertensive heart disease, but not following ischemic injury. Areas of interstitial fibrosis in ischemic and hypertensive hearts actively express Tcf21, Wt1, and Tbx18. In all areas of fibrosis, cells that express epicardial progenitor factors are distinct from CD45-positive immune cells. In human diseased hearts, differential expression of Tcf21, Wt1, and Tbx18 also is detected with epicardial, perivascular, and interstitial fibrosis, indicating conservation of reactivated developmental mechanisms in cardiac fibrosis in mice and humans. Together, these data provide evidence for distinct fibrogenic mechanisms that include Tcf21, separate from Wt1 and Tbx18, in different fibroblast populations in response to specific types of cardiac injury.

Placental growth factor regulates cardiac adaptation and hypertrophy through a paracrine mechanism.

RATIONALE: Paracrine growth factor-mediated crosstalk between cardiac myocytes and nonmyocytes in the heart is critical for programming adaptive cardiac hypertrophy in which myocyte size, capillary density, and the extracellular matrix function coordinately. OBJECTIVE: To examine the role that placental growth factor (PGF) plays in the heart as a paracrine regulator of cardiac adaptation to stress stimulation. METHODS AND RESULTS: PGF is induced in the heart after pressure-overload stimulation, where it is expressed in both myocytes and nonmyocytes. We generated cardiac-specific and adult inducible PGF-overexpressing transgenic mice and analyzed Pgf(-/-) mice to examine the role that this factor plays in cardiac disease and paracrine signaling. Although PGF transgenic mice did not have a baseline phenotype or a change in capillary density, they did exhibit a greater cardiac hypertrophic response, a greater increase in capillary density, and increased fibroblast content in the heart in response to pressure-overload stimulation. PGF transgenic mice showed a more adaptive type of cardiac growth that was protective against signs of failure with pressure overload and neuroendocrine stimulation. Antithetically, Pgf(-/-) mice rapidly died of heart failure within 1 week of pressure overload, they showed an inability to upregulate angiogenesis, and they showed significantly less fibroblast activity in the heart. Mechanistically, we show that PGF does not have a direct effect on cardiomyocytes but works through endothelial cells and fibroblasts by inducing capillary growth and fibroblast proliferation, which secondarily support greater cardiac hypertrophy through intermediate paracrine growth factors such as interleukin-6. CONCLUSIONS: PGF is a secreted factor that supports hypertrophy and cardiac function during pressure overload by affecting endothelial cells and fibroblasts that in turn stimulate and support the myocytes through additional paracrine factors.