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Chronic inflammation and tissue fibrosis are common stress responses that worsen organ function, yet the molecular mechanisms governing their crosstalk are poorly understood. In diseased organs, stress-induced changes in gene expression fuel maladaptive cell state transitions and pathological interaction between diverse cellular compartments. Although chronic fibroblast activation worsens dysfunction of lung, liver, kidney, and heart, and exacerbates many cancers, the stress-sensing mechanisms initiating the transcriptional activation of fibroblasts are not well understood. Here, we show that conditional deletion of the transcription co-activatorBrd4inCx3cr1-positive myeloid cells ameliorates heart failure and is associated with a dramatic reduction in fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancyin vivoinCx3cr1-positive cells identified a large enhancer proximal toInterleukin-1 beta (Il1b), and a series of CRISPR deletions revealed the precise stress-dependent regulatory element that controlled expression ofIl1bin disease. Secreted IL1B functioned non-cell autonomously to activate a p65/RELA-dependent enhancer near the transcription factorMEOX1, resulting in a profibrotic response in human cardiac fibroblasts.In vivo, antibody-mediated IL1B neutralization prevented stress-induced expression ofMEOX1, inhibited fibroblast activation, and improved cardiac function in heart failure. The elucidation of BRD4-dependent crosstalk between a specific immune cell subset and fibroblasts through IL1B provides new therapeutic strategies for heart disease and other disorders of chronic inflammation and maladaptive tissue remodeling.

Cardiovascular diseases and specifically heart failure (HF) impact global health and impose a significant economic burden on society. Despite current advances in standard of care, the risks for death and readmission of HF patients remain unacceptably high and new therapeutic strategies to limit HF progression are highly sought. In disease settings, persistent mechanical or neurohormonal stress to the myocardium triggers maladaptive cardiac remodelling, which alters cardiac function and structure at both the molecular and cellular levels. The progression and magnitude of maladaptive cardiac remodelling ultimately leads to the development of HF. Classical therapies for HF are largely protein-based and mostly are targeted to ameliorate the dysregulation of neuroendocrine pathways and halt adverse remodelling. More recently, investigation of novel molecular targets and the application of cellular therapies, epigenetic modifications, and regulatory RNAs has uncovered promising new avenues to address HF. In this review, we summarize the current knowledge on novel cellular and epigenetic therapies and focus on two non-coding RNA-based strategies that reached the phase of early clinical development to counteract cardiac remodelling and HF. The current status of the development of translating those novel therapies to clinical practice, limitations, and future perspectives are additionally discussed.

RationaleCardiomyocytes and fibroblasts in the heart communicate through both secreted growth factors as well as through sensing the structural properties of the extracellular matrix that each helps generate. Previous studies have shown that defects in fibroblast activity during disease stimulation result in altered cardiomyocyte hypertrophy, although the role that collagen might play in this communication is unknown.ObjectiveHere we investigated how type I collagen maturation and disease-responsive matrix expansion in the heart by cardiac fibroblasts impacts cardiac fibrosis and cardiomyocyte hypertrophy.Methods and ResultsWe generated and characterized Col1a2-/- mice using standard gene-targeting. Col1a2-/- mice were viable, although by young adulthood their hearts showed alterations in extracellular matrix mechanical properties, as well as an unanticipated activation of cardiac fibroblasts and induction of a progressive fibrotic response. This included increases in fibroblast number and a progressive cardiac hypertrophy, with reduced functional performance by 9 months. Col1a2-loxP targeted mice were also generated and crossed with the tamoxifen-inducible Postn-MerCreMer knock-in mice to delete the Col1a2 gene in myofibroblasts post-pressure overload injury, to more specifically implicate fibroblasts as effectors of cardiomyocyte hypertrophy in vivo. Opposite to the gradual induction of cardiac hypertrophy observed in germline Col1a2-/- mice as they matured developmentally, adult fibroblast-specific deletion of Col1a2 during pressure overload protected these mice from cardiac hypertrophy in the first week with a delayed fibrotic response. However, this reduction in hypertrophy due to myofibroblast-specific Col1a2 deletion was gradually lost over 2 and 6 weeks of pressure overload as augmented fibrosis returned.ConclusionsDefective type I collagen in the developing heart alters the structural integrity of the extracellular matrix that leads to fibroblast expansion, activation, fibrosis and hypertrophy with progressive cardiomyopathy in adulthood. However, acute deletion of type I collagen production for the first time in the adult heart during pressure overload prevents ECM expansion and inhibits cardiomyocyte hypertrophy, while gradual restoration of fibrosis again permitted hypertrophy comparable to controls.

Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient anti-fibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified mRNA in T cell targeted lipid nanoparticles. The efficacy of these in vivo reprogrammed CAR T cells was evaluated by injecting CD5-targeted lipid nanoparticles into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Anti-fibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA targeted lipid nanoparticles reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells holds promise as a therapeutic platform to treat various diseases.

During the postnatal period in mammals, the cardiac muscle transitions from hyperplasic to hypertrophic growth, the extracellular matrix (ECM) undergoes remodeling, and the heart loses regenerative capacity. While ECM maturation and crosstalk between cardiac fibroblasts (CFs) and cardiomyocytes (CMs) have been implicated in neonatal heart development, not much is known about specialized fibroblast heterogeneity and function in the early postnatal period. In order to better understand CF functions in heart maturation and postnatal cardiomyocyte cell-cycle arrest, we have performed gene expression profiling and ablation of postnatal CF populations. Fibroblast lineages expressing Tcf21 or Periostin were traced in transgenic GFP reporter mice, and their biological functions and transitions during the postnatal period were examined in sorted cells using RNA sequencing. Highly proliferative Periostin (Postn)+ lineage CFs were found from postnatal day 1 (P1) to P11 but were not detected at P30, due to a repression of Postn gene expression. This population was less abundant and transcriptionally different from Tcf21+ resident CFs. The specialized Postn+ population preferentially expresses genes related to cell proliferation and neuronal development, while Tcf21+ CFs differentially express genes related to ECM maturation at P7 and immune crosstalk at P30. Ablation of the Postn+ CFs from P0 to P6 led to altered cardiac sympathetic nerve patterning and a reduction in binucleation and hypertrophic growth with increased fetal troponin (TroponinI1) expression in CM. Thus, postnatal CFs are heterogeneous and include a transient proliferative Postn+ population required for cardiac nerve development and cardiomyocyte maturation soon after birth.

Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day1,2, despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biological effect3. The rationale for these cell therapy trials is derived from animal studies that show a modest but reproducible improvement in cardiac function in models of cardiac ischaemic injury4,5. Here we examine the mechanistic basis for cell therapy in mice after ischaemia-reperfusion injury, and find that-although heart function is enhanced-it is not associated with the production of new cardiomyocytes. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2+ and CX3CR1+ macrophages. Intracardiac injection of two distinct types of adult stem cells, cells killed by freezing and thawing or a chemical inducer of the innate immune response all induced a similar regional accumulation of CCR2+ and CX3CR1+ macrophages, and provided functional rejuvenation to the heart after ischaemia-reperfusion injury. This selective macrophage response altered the activity of cardiac fibroblasts, reduced the extracellular matrix content in the border zone and enhanced the mechanical properties of the injured area. The functional benefit of cardiac cell therapy is thus due to an acute inflammatory-based wound-healing response that rejuvenates the infarcted area of the heart.

Background: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy. Methods: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1 WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1 WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1 WT/S52F mice and determine its effects on PH and RV hypertrophy. Results: Foxf1 WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1 WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1 WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1 WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling. Conclusions: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.

S-palmitoylation is a reversible lipid modification that regulates trafficking, localization, activity, and/or stability of protein substrates by serving as a fatty acid anchor to cell membranes. However, S-palmitoylation-dependent control of signal transduction in cardiomyocytes and its effects on cardiac physiology are not well understood. We performed an in vivo gain-of-function screen of zinc finger Asp-His-His-Cys (zDHHC) family S-acyl transferases that catalyze S-palmitoylation and identified the Golgi-localized enzyme zDHHC3 as a critical regulator of cardiac maladaptation. The closely-related enzyme, zDHHC7, also induced severe cardiomyopathy but this effect was not observed with overexpression of plasma membrane enzyme zDHHC5, endoplasmic reticulum enzyme zDHHC6, or Golgi enzyme zDHHC13. To identify effectors that may underlie zDHHC3-induced cardiomyopathy we performed quantitative site-specific S-acyl proteomics in zDHHC3-overexpressing cells that revealed the small GTPase Rac1 as a novel substrate. We generated cardiomyocyte-specific transgenic mice overexpressing zDHHC3, which develop severe cardiac disease. Cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice are preceded by enhanced S-palmitoylation of Rac1 and induction of additional Rho family small GTPases including RhoA, Cdc42, and the Rho family-specific chaperone RhoGDI. In contrast, transgenic mice overexpressing an enzymatically-dead mutant of zDHHC3 do not exhibit this profound induction of RhoGTPase signaling or develop cardiac disease. Rac1 S-palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts. Taken together, these data suggest inhibition of zDHHC3/7 S-acyl transferase activity at the cardiomyocyte Golgi or disruption of Rac1 S-palmitoylation as novel therapeutic strategies to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.

Background: The adult mammalian heart displays a cardiomyocyte turnover rate of ≈1%/y throughout postnatal life and after injuries such as myocardial infarction (MI), but the question of which cell types drive this low level of new cardiomyocyte formation remains contentious. Cardiac-resident stem cells marked by stem cell antigen-1 (Sca-1, gene name Ly6a ) have been proposed as an important source of cardiomyocyte renewal. However, the in vivo contribution of endogenous Sca-1 + cells to the heart at baseline or after MI has not been investigated. Methods: Here we generated Ly6a gene–targeted mice containing either a constitutive or an inducible Cre recombinase to perform genetic lineage tracing of Sca-1 + cells in vivo. Results: We observed that the contribution of endogenous Sca-1 + cells to the cardiomyocyte population in the heart was + cells abundantly contributed to the cardiac vasculature in mice during physiological growth and in the post-MI heart during cardiac remodeling. Specifically, Sca-1 lineage-traced endothelial cells expanded postnatally in the mouse heart after birth and into adulthood. Moreover, pulse labeling of Sca-1 + cells with an inducible Ly6a -MerCreMer allele also revealed a preferential expansion of Sca-1 lineage-traced endothelial cells after MI injury in the mouse. Conclusions: Cardiac-resident Sca-1 + cells are not significant contributors to cardiomyocyte renewal in vivo. However, cardiac Sca-1 + cells represent a subset of vascular endothelial cells that expand postnatally with enhanced responsiveness to pathological stress in vivo.

BACKGROUND: Myeloid cells play an important role in a wide variety of cardiovascular disorders, including both ischemic and non-ischemic cardiomyopathies. Neuregulin-1 (NRG-1)/ErbB signaling has recently emerged as an important factor contributing to the control of inflammatory activation of myeloid cells after an ischemic injury. However, the role of ErbB signaling in myeloid cells in non-ischemic cardiomyopathy is not fully understood. This study investigated the role of ErbB3 receptors in the regulation of early adaptive response using a mouse model of transverse aortic constriction (TAC) for non-ischemic cardiomyopathy. METHODS AND RESULTS: TAC surgery was performed in groups of age- and sex-matched myeloid cell-specific ErbB3-deficient mice (ErbB3(MyeKO)) and control animals (ErbB3(MyeWT)). The number of cardiac CD45 immune cells, CD11b myeloid cells, Ly6G neutrophils, and Ly6C monocytes was determined using flow cytometric analysis. Five days after TAC, survival was dramatically reduced in male but not female ErbB3(MyeKO) mice or control animals. The examination of lung weight to body weight ratio suggested that acute pulmonary edema was present in ErbB3(MyeKO) male mice after TAC. To determine the cellular and molecular mechanisms involved in the increased mortality in ErbB3(MyeKO) male mice, cardiac cell populations were examined at day 3 post-TAC using flow cytometry. Myeloid cells accumulated in control but not in ErbB3(MyeKO) male mouse hearts. This was accompanied by increased proliferation of Sca-1 positive non-immune cells (endothelial cells and fibroblasts) in control but not ErbB3(MyeKO) male mice. No significant differences in intramyocardial accumulation of myeloid cells or proliferation of Sca-1 cells were found between the groups of ErbB3(MyeKO) and ErbB3(MyeWT) female mice. An antibody-based protein array analysis revealed that IGF-1 expression was significantly downregulated only in ErbB3(MyeKO) mice hearts compared to control animals after TAC. CONCLUSION: Our data demonstrate the crucial role of myeloid cell-specific ErbB3 signaling in the cardiac accumulation of myeloid cells, which contributes to the activation of cardiac endothelial cells and fibroblasts and development of an early adaptive response to cardiac pressure overload in male mice.

Background: Myxomatous valve degeneration (MVD) involves the progressive thickening and degeneration of the heart valves, leading to valve prolapse, regurgitant blood flow, and impaired cardiac function. Leukocytes composed primarily of macrophages have recently been detected in myxomatous valves, but the timing of the presence and the contributions of these cells in MVD progression are not known. Methods: We examined MVD progression, macrophages, and the valve microenvironment in the context of Marfan syndrome (MFS) using mitral valves from MFS mice ( Fbn1 C1039G/+ ), gene-edited MFS pigs ( FBN1 Glu433AsnfsX98/+ ), and patients with MFS. Additional histological and transcriptomic evaluation was performed by using nonsyndromic human and canine myxomatous valves, respectively. Macrophage ontogeny was determined using MFS mice transplanted with mTomato+ bone marrow or MFS mice harboring RFP (red fluorescent protein)–tagged C-C chemokine receptor type 2 (CCR2) monocytes. Mice deficient in recruited macrophages ( Fbn1 C1039G/+ ;Ccr2 RFP/RFP ) were generated to determine the requirements of recruited macrophages to MVD progression. Results: MFS mice recapitulated histopathological features of myxomatous valve disease by 2 months of age, including mitral valve thickening, increased leaflet cellularity, and extracellular matrix abnormalities characterized by proteoglycan accumulation and collagen fragmentation. Diseased mitral valves of MFS mice concurrently exhibited a marked increase of infiltrating (MHCII+, CCR2+) and resident macrophages (CD206+, CCR2–), along with increased chemokine activity and inflammatory extracellular matrix modification. Likewise, mitral valve specimens obtained from gene-edited MFS pigs and human patients with MFS exhibited increased monocytes and macrophages (CD14+, CD64+, CD68+, CD163+) detected by immunofluorescence. In addition, comparative transcriptomic evaluation of both genetic (MFS mice) and acquired forms of MVD (humans and dogs) unveiled a shared upregulated inflammatory response in diseased valves. Remarkably, the deficiency of monocytes was protective against MVD progression, resulting in a significant reduction of MHCII macrophages, minimal leaflet thickening, and preserved mitral valve integrity. Conclusions: All together, our results suggest sterile inflammation as a novel paradigm to disease progression, and we identify, for the first time, monocytes as a viable candidate for targeted therapy in MVD.

Satellite cells are skeletal muscle resident stem cells that regenerate adult myofibers following an acute injury to muscle. Despite the assumption that the loss of satellite cells would be detrimental in a chronic regeneration-inducing muscle disease such as muscular dystrophy, this assumption has never been tested using mouse genetics. Here we generated a novel model of satellite cell ablation and crossed it with mouse models of muscular dystrophy to directly investigate how critical these cells are in maintaining muscle during a chronic degenerative disorder. Satellite cell deletion in 2-week-old young dystrophic mice provided noticeable improvements in histopathology and function, although at this early timepoint it was utimately detrimental because muscle size was not sufficient to permit survival. However, depletion of satellite cells beginning at 2 months of age in dystrophic mice provided similar histological and functional improvements but without compromising muscle size. The improved profile showed fewer damaged fibers, less myofiber central nucleation, increased sarcolemma integrity, decreased fibrosis and a dramatic size increase in the remaining myofibers. At the functional level, young adult dystrophic mice lacking satellite cells performed significantly better than those with satellite cells when exercised on a treadmill. Thus, loss of satellite cells during early adulthood in dystrophic mice produces an unexpected protective effect.

Death of adult cardiac myocytes and supportive tissues resulting from cardiovascular diseases such as myocardial infarction (MI) is the proximal driver of pathological ventricular remodeling that often culminates in heart failure. After MI the heart may lose up to a billion myocytes(1), resulting in impaired pump function, persistent sympathetic neurohormonal activation, pathological hypertrophy of surviving myocytes, and replacement fibrosis(2). Despite advances in the diagnosis and early treatment of acute MI, the lifetime risk and overall health burden from heart failure remain profoundly high(3). This is partially because no currently available therapeutic, barring heart transplantation, can directly replenish myocytes lost from the injured heart. For decades, the field has struggled to define the intrinsic capacity and cellular sources for endogenous myocyte turnover in pursuing more innovative therapeutic strategies aimed at regenerating the injured heart. While controversy persists to this day as to the best therapeutic regenerative strategy to employ, a growing consensus has been reached that the very limited capacity for new myocyte formation in the adult mammalian heart is due to proliferation of existing cardiac myocytes, but not due to the activity of an endogenous progenitor cell source of some sort. Hence, future therapeutic approaches should take into consideration the fundamental biology of myocyte renewal in designing strategies to potentially replenish these cells from the injured heart.

The annual Basic Cardiovascular Sciences (BCVS) Scientific Sessions remains a premier conference destination for those in the basic and translational cardiovascular research community. This year’s BCVS meeting was held at the downtown waterfront in Boston, MA from July 29 to August 1, 2019. To say that BCVS 2019 was well-attended is truly an understatement, as this year’s meeting broke all BCVS conference records with over 1000 registered attendees, over 600 posters, and a plethora of new offerings, such as expanded early career sessions and the first Asian Cardiovascular Symposium. Here, we highlight some of the emerging science and exciting programming that was featured at the 2019 BCVS Scientific Sessions.

The identity and function of the fibroblast, a highly prevalent cell type in the heart, have remained poorly defined. Recent faithful genetic lineage–tracing studies revealed that during development, the cardiac fibroblasts are derived from the epicardium and the endothelium, whereas in the adult heart, they constitute the cardiac injury–responsive resident fibroblast. In the current issue of the JCI, Molkentin and colleagues decipher the time course and mechanism of the fibroblast in response to myocardial infarction (MI). The model they propose is surprisingly simple and clear. It consists of three major phases. First, fibroblasts in the ischemic area die. Second, surrounding fibroblasts proliferate and migrate into the spaces created by dying cardiomyocytes over a few days. The new fibroblasts in the scar are activated and adopt a smooth muscle actin– and periostin-positive “myofibroblast” phenotype, which appears to last as long as the scar is not mature (~10 days after MI). In the third phase, initially proliferating myofibroblasts lose smooth muscle actin expression and convert to a nonproliferating, matrix-producing phenotype with a newly acquired tendon gene signature. Interestingly, this state appears to differ from that of quiescent fibroblasts in the uninjured heart, as it is resistant to proliferative stimuli. These cells are therefore termed “matrifibrocytes,” a novel category whose study will certainly further advance the field.

This article contains errors in Figs. 5 and 6, for which we apologize. In Fig. 5f, the image ‘E12.5 tail’ was inadvertently replaced with a duplicate of the image ‘E12.5 trunk’ from the same panel. In Figure 6d, the image ‘E9.5/OH-TAM E8.5, embryo’ was inadvertently replaced with a duplicate of the image ‘E10.5/ OH-TAM E8.5, embryo’ from Fig. 6b. The corrected versions of these figures appear in the Author Correction associated with this Article.

Collagen production in the adult heart is thought to be regulated by the fibroblast, although cardiomyocytes and endothelial cells also express multiple collagen mRNAs. Molecular chaperones are required for procollagen biosynthesis, including heat shock protein 47 (Hsp47). To determine the cell types critically involved in cardiac injury–induced fibrosis the Hsp47 gene was deleted in cardiomyocytes, endothelial cells, or myofibroblasts. Deletion of Hsp47 from cardiomyocytes during embryonic development or adult stages, or deletion from adult endothelial cells, did not affect cardiac fibrosis after pressure overload injury. However, myofibroblast-specific ablation of Hsp47 blocked fibrosis and deposition of collagens type I, III, and V following pressure overload as well as significantly reduced cardiac hypertrophy. Fibroblast-specific Hsp47-deleted mice showed lethality after myocardial infarction injury, with ineffective scar formation and ventricular wall rupture. Similarly, only myofibroblast-specific deletion of Hsp47 reduced fibrosis and disease in skeletal muscle in a mouse model of muscular dystrophy. Mechanistically, deletion of Hsp47 from myofibroblasts reduced mRNA expression of fibrillar collagens and attenuated their proliferation in the heart without affecting paracrine secretory activity of these cells. The results show that myofibroblasts are the primary mediators of tissue fibrosis and scar formation in the injured adult heart, which unexpectedly affects cardiomyocyte hypertrophy., Deletion of the Hsp47 gene in cardiac myofibroblasts, but not myocytes or endothelial cells, selectively inhibits fibrosis in mice following injury.

S-palmitoylation is a reversible lipid modification that regulates protein trafficking, localization, and function by providing a lipid tag that serves as a molecular instruction for association wi...

The concept that cardiac-derived cKit+ cells can regenerate the injured adult heart by transdifferentiating into functioning cardiomyocytes was proposed 14 years ago although it remains controversial because of negative data from multiple independent laboratories. Irreproducibility of cardiac cKit+ cell studies was attributed to the differences in cell isolation, selection, and expansion before in vivo application. This Viewpoint will discuss recent results that again change the formula for how cardiac cKit+ cells must be isolated and processed to be cardiomyogenic, as well as discuss the uncertain in vivo relevance of cKit+ cells as putative cardiomyocyte-producing stem cells. The regenerative and myogenic potential of cKit+ cardiac-resident stem cells (CSCs) remains a dominant and contentious topic. In vitro expanded cKit+ CSCs were originally described 14 years ago as a source for robust new myocyte generation when injected directly into the adult rodent heart after myocardial infarction,1 and although some laboratories have confirmed the basic principle, the magnitude of the effect remains unclear.2 More recently, Ellison et al3 showed that a clonal cKit+ cell line derived from a population of cardiac CD45− cKit+ cells was capable of widely regenerating isoproterenol-injured adult rodent hearts with abundant new cardiomyocytes, after simple tail vein infusion. However, many independent laboratories reported that adult cardiac-derived cKit+ cells did not generate appreciable new cardiomyocytes when directly injected back into the injured rat or mouse heart.4–7 In addition to controversies surrounding the regenerative potential of injected cKit+ cell formulations, until recently the field has not attempted to directly address if endogenous cardiac cKit+ cells are true cardiomyocyte producing stem cells of meaningful significance.8 …

Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day1–3 despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biologic effect4–6. The rationale for these cell therapy trials is derived from animal studies that show a modest but reproducible improvement in cardiac function in models of cardiac ischemic injury7–9. Here we examined the mechanistic basis for cell therapy in mice after ischemia/reperfusion (I/R) injury, and while heart function was enhanced, it was not associated with new cardiomyocyte production. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2+ and CX3CR1+ macrophages. Here we observed that intra-cardiac injection of 2 distinct types of progenitor cells, freeze/thaw-killed cells or a chemical inducer of the innate immune response similarly induced regional CCR2+ and CX3CR1+ macrophage accumulation and provided functional rejuvenation to the I/R-injured heart. Mechanistically, this selective macrophage response altered cardiac fibroblast activity and reduced border zone extracellular matrix (ECM) content and enhanced the mechanical properties of the injured area. The functional benefit of cardiac cell therapy is thus due to an acute inflammatory-based wound healing response that rejuvenates the mechanical properties of the infarcted area of the heart. Such results suggest a re-evaluation of strategies underlying cardiac cell therapy in current and planned human clinical trials.

Objective— Periostin is a secreted protein that can alter extracellular matrix remodeling in response to tissue injury. However, the functional role of periostin in the development of atherosclerotic plaques has yet to be described despite its observed induction in diseased vessels and presence in the serum. Approach and Results— Hyperlipidemic, apolipoprotein E–null mice ( ApoE −/ − ) were crossed with periostin ( Postn −/− ) gene–deleted mice and placed on a high-fat diet for 6 or 14 weeks to induce atherosclerosis. En face analysis of aortas showed significantly decreased lesion areas of ApoE −/− Postn −/− mice compared with ApoE −/− mice, as well as a reduced inflammatory response with less macrophage content. Moreover, diseased aortas from ApoE −/− Postn −/− mice displayed a disorganized extracellular matrix with less collagen cross linking and smaller fibrotic caps, as well as increased matrix metalloproteinase-2, metalloproteinase-13, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase-1 mRNA expression. Furthermore, the loss of periostin was associated with a switch in vascular smooth muscle cells toward a more proliferative and synthetic phenotype. Mechanistically, the loss of periostin reduced macrophage recruitment by transforming growth factor-β in cellular migration assays. Conclusions— These are the first genetic data detailing the function of periostin as a regulator of atherosclerotic lesion formation and progression. The data suggest that periostin could be a therapeutic target for atherosclerotic plaque formation through modulation of the immune response and extracellular matrix remodeling.

There are 3 protein phosphatase 1 (PP1) catalytic isoforms (α, β and γ) encoded within the mammalian genome. These 3 gene products share ~90% amino acid homology within their catalytic domains but each has unique N- and C-termini that likely underlie distinctive subcellular localization or functionality. In this study, we assessed the effect associated with the loss of each PP1 isoform in the heart using a conditional Cre-loxP targeting approach in mice. Ppp1ca-loxP, Ppp1cb-loxP and Ppp1cc-loxP alleles were crossed with either an Nkx2.5-Cre knock-in containing allele for early embryonic deletion or a tamoxifen inducible α-myosin heavy chain (αMHC)-MerCreMer transgene for adult and cardiac-specific deletion. We determined that while deletion of Ppp1ca (PP1α) or Ppp1cc (PP1γ) had little effect on the whole heart, deletion of Ppp1cb (PP1β) resulted in concentric remodeling of the heart, interstitial fibrosis and contractile dysregulation, using either the embryonic or adult-specific Cre-expressing alleles. However, myocytes isolated from Ppp1cb deleted hearts surprisingly showed enhanced contractility. Mechanistically we found that deletion of any of the 3 PP1 gene-encoding isoforms had no effect on phosphorylation of phospholamban, nor were Ca(2+) handling dynamics altered in adult myocytes from Ppp1cb deleted hearts. However, the loss of Ppp1cb from the heart, but not Ppp1ca or Ppp1cc, resulted in elevated phosphorylation of myofilament proteins such as myosin light chain 2 and cardiac myosin binding protein C, consistent with an enriched localization profile of this isoform to the sarcomeres. These results suggest a unique functional role for the PP1β isoform in affecting cardiac contractile function.

Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day

The master cytokine TGF-β mediates tissue fibrosis associated with inflammation and tissue injury. TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-β-Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload-induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-β receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload-induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-β mutant transgene. Interestingly, cardiac fibroblast-specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-β-Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.

BackgroundInjury due to myocardial infarction (MI) is largely irreversible. Once an infarct has occurred, the clinical goal becomes limiting remodeling, preserving left ventricular function, and preventing heart failure. Although traditional approaches (e.g., β-blockers) partially preserve left ventricular function, novel strategies are needed to limit ventricular remodeling post-MI.ObjectivesThe aim of this study was to determine the role of glycogen synthase kinase–3α (GSK-3α) in post-MI remodeling.MethodsMice with cardiomyocyte-specific conditional deletion of Gsk3α and littermate controls underwent sham or MI surgery. Heart function was assessed using serial M-mode echocardiography.ResultsGsk3α deletion in the heart markedly limits remodeling and preserves left ventricular function post-MI. This is due at least in part to dramatic thinning and expansion of the scar in the control hearts, which was less in the heart of knockout (KO) mice. In contrast, the border zone in the KO mice demonstrated a much thicker scar, and there were more viable cardiomyocytes within the scar/border zone. This was associated with less apoptosis and more proliferation of cardiomyocytes in the KO mice. Mechanistically, reduced apoptosis was due, at least in part, to a marked decrease in the Bax/Bcl-2 ratio, and increased cardiomyocyte proliferation was mediated through cyclin E1 and E2F-1 in the hearts of the KO mice.ConclusionsTaken together, these findings show that reducing GSK-3α expression in cardiomyocytes limits ventricular remodeling and preserves cardiac function post-MI. Specifically targeting GSK-3α could be a novel strategy to limit adverse remodeling and heart failure.

Background— Myocardial infarction–induced remodeling includes chamber dilatation, contractile dysfunction, and fibrosis. Of these, fibrosis is the least understood. After myocardial infarction, activated cardiac fibroblasts deposit extracellular matrix. Current therapies to prevent fibrosis are inadequate, and new molecular targets are needed. Methods and Results— Herein we report that glycogen synthase kinase-3β (GSK-3β) is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using 2 fibroblast-specific GSK-3β knockout mouse models, we show that deletion of GSK-3β in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Deletion of GSK-3β induces a profibrotic myofibroblast phenotype in isolated cardiac fibroblasts, in post–myocardial infarction hearts, and in mouse embryonic fibroblasts deleted for GSK-3β. Mechanistically, GSK-3β inhibits profibrotic transforming growth factor-β1/SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3β resulted in the significant increase of SMAD-3 transcriptional activity. This pathway is central to the pathology because a small-molecule inhibitor of SMAD-3 largely prevented fibrosis and limited left ventricular remodeling. Conclusions— These studies support targeting GSK-3β in myocardial fibrotic disorders and establish critical roles of cardiac fibroblasts in remodeling and ventricular dysfunction.

Retinal pigment epithelial cells of teleosts contain numerous melanosomes (pigment granules) that exhibit light-dependent motility. In light, melanosomes disperse out of the retinal pigment epithelium (RPE) cell body (CB) into long apical projections that interdigitate with rod photoreceptors, thus shielding the photoreceptors from bleaching. In darkness, melanosomes aggregate through the apical projections back into the CB. Previous research has demonstrated that melanosome motility in the RPE CB requires microtubules, but in the RPE apical projections, actin filaments are necessary and sufficient for motility. We used myosin S1 labeling and platinum replica shadowing of dissociated RPE cells to determine actin filament polarity in apical projections. Actin filament bundles within RPE apical projections are uniformly oriented with barbed ends toward the distal tips. Treatment of RPE cells with the tetravalent lectin, Concanavalin A, which has been shown to suppress cortical actin flow by crosslinking of cell-surface proteins, inhibited melanosome aggregation and stimulated ectopic filopodia formation but did not block melanosome dispersion. The polarity orientation of F-actin in apical projections suggests that a barbed-end directed myosin motor could effect dispersion of melanosomes from the CB into apical projections. Inhibition of aggregation, but not dispersion, by ConA confirms that different actin-dependent mechanisms control these two processes and suggests that melanosome aggregation is sensitive to treatments previously shown to disrupt actin cortical flow.

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in aging. We also observed severe vacuolar degeneration of myofibers and large tubular aggregates in skeletal muscle, consistent with impaired clearance of insoluble cellular debris. Other organ systems, including gut, liver, and the skeletal system, also demonstrated age-related pathologies. Mechanistically, we found marked activation of mTORC1 and associated suppression of autophagy markers in KO mice. Loss of GSK-3α, either by pharmacologic inhibition or Gsk3a gene deletion, suppressed autophagy in fibroblasts. mTOR inhibition rescued this effect and reversed the established pathologies in the striated muscle of the KO mouse. Thus, GSK-3α is a critical regulator of mTORC1, autophagy, and aging. In its absence, aging/senescence is accelerated in multiple tissues. Strategies to maintain GSK-3α activity and/or inhibit mTOR in the elderly could retard the appearance of age-related pathologies.

Rationale: Catecholamines increase cardiac contractility, but exposure to high concentrations or prolonged exposures can cause cardiac injury. A recent study demonstrated that a single subcutaneous injection of isoproterenol (ISO; 200 mg/kg) in mice causes acute myocyte death (8%–10%) with complete cardiac repair within a month. Cardiac regeneration was via endogenous cKit + cardiac stem cell–mediated new myocyte formation. Objective: Our goal was to validate this simple injury/regeneration system and use it to study the biology of newly forming adult cardiac myocytes. Methods and Results: C57BL/6 mice (n=173) were treated with single injections of vehicle, 200 or 300 mg/kg ISO, or 2 daily doses of 200 mg/kg ISO for 6 days. Echocardiography revealed transiently increased systolic function and unaltered diastolic function 1 day after single ISO injection. Single ISO injections also caused membrane injury in ≈10% of myocytes, but few of these myocytes appeared to be necrotic. Circulating troponin I levels after ISO were elevated, further documenting myocyte damage. However, myocyte apoptosis was not increased after ISO injury. Heart weight to body weight ratio and fibrosis were also not altered 28 days after ISO injection. Single- or multiple-dose ISO injury was not associated with an increase in the percentage of 5-ethynyl-2′-deoxyuridine–labeled myocytes. Furthermore, ISO injections did not increase new myocytes in cKit +/Cre ×R-GFP transgenic mice. Conclusions: A single dose of ISO causes injury in ≈10% of the cardiomyocytes. However, most of these myocytes seem to recover and do not elicit cKit + cardiac stem cell–derived myocyte regeneration.

Background— The molecular pathways that regulate the extent of ischemic injury and post–myocardial infarction (MI) remodeling are not well understood. We recently demonstrated that glycogen synthase kinase-3α (GSK-3α) is critical to the heart's response to pressure overload. However, the role, if any, of GSK-3α in regulating ischemic injury and its consequences is not known. Methods and Results— MI was induced in wild-type (WT) versus GSK-3α (−/−) (KO) littermates by left anterior descending coronary artery ligation. Pre-MI, WT, and KO hearts had comparable chamber dimensions and ventricular function, but as early as 1 week post-MI, KO mice had significantly more left ventricular dilatation and dysfunction than WT mice. KO mice also had increased mortality during the first 10 days post-MI (43% versus 22%; P =0.04), and postmortem examination confirmed cardiac rupture as the cause of most of the deaths. In the mice that survived the first 10 days, left ventricular dilatation and dysfunction remained worse in the KO mice throughout the study (8 weeks). Hypertrophy, fibrosis, and heart failure were all increased in the KO mice. Given the early deaths due to rupture and the significant reduction in left ventricular function evident as early as 1 week post-MI, we examined infarct size following a 48-hour coronary artery ligation and found it to be increased in the KO mice. This was accompanied by increased apoptosis in the border zone of the MI. This increased susceptibility to ischemic injury–induced apoptosis was also seen in cardiomyocytes isolated from the KO mice that were exposed to hypoxia. Finally, Bax translocation to the mitochondria and cytochrome C release into the cytosol were increased in the KO mice. Conclusion— GSK-3α confers resistance to ischemic injury, at least in part, via limiting apoptosis. Loss of GSK-3α promotes ischemic injury, increases risk of cardiac rupture, accentuates post-MI remodeling and left ventricular dysfunction, and increases the progression to heart failure. These findings are in striking contrast to multiple previous reports in which deletion or inhibition of GSK-3β is protective.

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.

Rationale : Numerous studies have proposed that glycogen synthase kinase (GSK)-3β is a central regulator of the hypertrophic response of cardiomyocytes. However, all of this work has relied on overexpression of GSK-3β, expression of constitutively active mutants, or small molecule inhibitors with documented off-target effects. Genetic loss of function approaches have not been used in the adult mouse because germ-line deletion of GSK-3β is embryonic-lethal. Objective : This study was designed to define the role played by GSK-3β in pressure overload (PO)-induced hypertrophy and remodeling following myocardial infarction (MI). Methods and Results : We used a mouse model that allows inducible, cardiomyocyte-specific deletion of GSK-3β in the adult knockout. Surprisingly, we find that knockout mice exposed to PO induced by thoracic aortic constriction exhibit a normal hypertrophic response. Thus, in contrast to virtually all prior published studies, GSK-3β appears to play at most a minor role in the hypertrophic response to PO stress. However, GSK-3β does regulate post-MI remodeling because the GSK-3β knockouts had less left ventricular dilatation and better-preserved left ventricular function at up to 8 weeks post-MI despite demonstrating significantly more hypertrophy in the remote myocardium. Deletion of GSK-3β also led to increased cardiomyocyte proliferation following PO and MI. Conclusions : Deletion of GSK-3β protects against post-MI remodeling and promotes stress-induced cardiomyocyte proliferation in the adult heart. These studies suggest that inhibition of GSK-3β could be a strategy to both prevent remodeling and to promote cardiac regeneration in pathological states.

Multiple studies have shown an innate capacity of the adult mammalian heart to partially regenerate following stress or injuries such as myocardial infarction (MI). However, this modest capacity is insufficient to resolve the significant damage that occurs post-MI, thus efforts have centered on bolstering this regenerative potential by delivery of various stem or progenitor cell types. To-date, over fifty clinical trials have evaluated cell-based therapeutics to induce regeneration in the setting of ischemic heart disease - mostly using bone marrow mononuclear cells (BM-MNCs) although c-Kit + cardiac progenitor cells (CPCs) and mesenchymal stromal cells (MSCs) from bone were also evaluated. Collectively, cell therapy has demonstrated clinical safety; yet overall effectiveness has been questioned, as evidence that transplanted cells differentiate into cardiomyocytes is lacking. Hence, studies showing some functional benefit have ascribed it to enhanced endogenous regeneration via paracrine factors secreted by the implanted cells. One hypothesis is that these factors stimulate resident c-Kit + progenitors to generate new myocardium. We recently developed an inducible genetic system to trace endogenous c-Kit + cells in the murine heart. We now use this system to evaluate whether delivery of different progenitor-like cell types can enhance c-Kit + -derived myocyte or vessel formation in the uninjured heart; using a dual-reporter strategy to differentially fate map transplanted cells versus endogenous c-Kit + cells. We observed that BM-MNCs induced a modest increase in c-Kit + -derived, CD31 + vessels proximal to the site of injection, although no new myocytes were observed. There was no evidence for fusion between BM-MNCs and host myocardium, or for prolonged engraftment, as by 6 weeks virtually all injected cells were cleared. Taken together, this suggests that BM-MNCs are capable of inducing an acute c-Kit + -mediated vascular response in the uninjured heart. We are also currently evaluating CPCs, bone marrow or adipose-derived MSCs, and bone stromal cells for their capacity to stimulate c-Kit + cells, either without injury or after MI. Our goal is to better define the paracrine hypothesis, improving cell-based therapies for application in humans.

Myocardial ischemia results in death of cardiac myocytes via tightly regulated and interconnected signaling pathways. Protein kinases play crucial roles in this regulation and are highly amenable to therapeutic intervention, making targeted inhibition an attractive strategy for ischemic heart disease. Recent studies have uncovered numerous kinases that participate in the cardiomyocyte response to ischemic injury, thus potentiating the development of new therapeutics. Moreover, many kinase signaling pathways converge at the mitochondria, a key participant in both cardiomyocyte physiology and the pathogenesis of ischemic heart disease. Herein we highlight kinase pathways regulating three major drivers of cell death: mitochondrial permeability transition pore opening (mPTP), programmed necrosis and Ca2+ overload-induced mitochondrial dysfunction. Inhibition of each of these kinase pathways has been proposed as a means to limit cardiomyocyte death from ischemia/reperfusion (I/R) injury.

Rationale: Sorafenib is an effective treatment for renal cell carcinoma, but recent clinical reports have documented its cardiotoxicity through an unknown mechanism. Objective: Determining the mechanism of sorafenib-mediated cardiotoxicity. Methods and Results: Mice treated with sorafenib or vehicle for 3 weeks underwent induced myocardial infarction (MI) after 1 week of treatment. Sorafenib markedly decreased 2-week survival relative to vehicle-treated controls, but echocardiography at 1 and 2 weeks post MI detected no differences in cardiac function. Sorafenib-treated hearts had significantly smaller diastolic and systolic volumes and reduced heart weights. High doses of sorafenib induced necrotic death of isolated myocytes in vitro, but lower doses did not induce myocyte death or affect inotropy. Histological analysis documented increased myocyte cross-sectional area despite smaller heart sizes after sorafenib treatment, further suggesting myocyte loss. Sorafenib caused apoptotic cell death of cardiac- and bone-derived c-kit+ stem cells in vitro and decreased the number of BrdU+ (5-bromo-2’-deoxyuridine+) myocytes detected at the infarct border zone in fixed tissues. Sorafenib had no effect on infarct size, fibrosis, or post-MI neovascularization. When sorafenib-treated animals received metoprolol treatment post MI, the sorafenib-induced increase in post-MI mortality was eliminated, cardiac function was improved, and myocyte loss was ameliorated. Conclusions: Sorafenib cardiotoxicity results from myocyte necrosis rather than from any direct effect on myocyte function. Surviving myocytes undergo pathological hypertrophy. Inhibition of c-kit+ stem cell proliferation by inducing apoptosis exacerbates damage by decreasing endogenous cardiac repair. In the setting of MI, which also causes large-scale cell loss, sorafenib cardiotoxicity dramatically increases mortality.

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.