Marjorie Maillet

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.

Cardiac-Specific Deletion of Gata4 Reveals Its Requirement for Hypertrophy, Compensation, and Myocyte Viability

The transcription factor GATA4 is a critical regulator of cardiac gene expression where it controls embryonic development, cardiomyocyte differentiation, and stress responsiveness of the adult heart. Traditional deletion of Gata4 caused embryonic lethality associated with endoderm defects and cardiac malformations, precluding an analysis of the role of GATA4 in the adult myocardium. To address the function of GATA4 in the adult heart, Gata4-loxP–targeted mice (Gata4fl/fl) were crossed with mice containing a β-myosin heavy chain (β-MHC) or α-MHC promoter-driven Cre transgene, which produced viable mice that survived into adulthood despite a 95% and 70% loss of GATA4 protein, respectively. However, cardiac-specific deletion of Gata4 resulted in a progressive and dosage-dependent deterioration in cardiac function and dilation in adulthood. Moreover, pressure overload stimulation induced rapid decompensation and heart failure in cardiac-specific Gata4-deleted mice. More provocatively, Gata4-deleted mice were compromised in their ability to hypertrophy following pressure overload or exercise stimulation. Mechanistically, cardiac-specific deletion of Gata4 increased cardiomyocyte TUNEL at baseline in embryos and adults as they aged, as well as dramatically increased TUNEL following pressure overload stimulation. Examination of gene expression profiles in the heart revealed a number of profound alterations in known GATA4-regulated structural genes as well as genes with apoptotic implications. Thus, GATA4 is a necessary regulator of cardiac gene expression, hypertrophy, stress-compensation, and myocyte viability.

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.

Signaling effectors underlying pathologic growth and remodeling of the heart

Cardiovascular disease is the number one cause of mortality in the Western world. The heart responds to many cardiopathological conditions with hypertrophic growth by enlarging individual myocytes to augment cardiac pump function and decrease ventricular wall tension. Initially, such cardiac hypertrophic growth is often compensatory, but as time progresses these changes become maladaptive. Cardiac hypertrophy is the strongest predictor for the development of heart failure, arrhythmia, and sudden death. Here we discuss therapeutic avenues emerging from molecular and genetic studies of cardiovascular disease in animal models. The majority of these are based on intracellular signaling pathways considered central to pathologic cardiac remodeling and hypertrophy, which then leads to heart failure. We focus our discussion on selected therapeutic targets that have more recently emerged and have a tangible translational potential given the available pharmacologic agents that could be readily evaluated in human clinical trials.

Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism

Muscular dystrophy is a general term encompassing muscle disorders that cause weakness and wasting, typically leading to premature death. Membrane instability, as a result of a genetic disruption within the dystrophin-glycoprotein complex (DGC), is thought to induce myofiber degeneration, although the downstream mechanism whereby membrane fragility leads to disease remains controversial. One potential mechanism that has yet to be definitively proven in vivo is that unregulated calcium influx initiates disease in dystrophic myofibers. Here we demonstrate that calcium itself is sufficient to cause a dystrophic phenotype in skeletal muscle independent of membrane fragility. For example, overexpression of transient receptor potential canonical 3 (TRPC3) and the associated increase in calcium influx resulted in a phenotype of muscular dystrophy nearly identical to that observed in DGC-lacking dystrophic disease models, including a highly similar molecular signature of gene expression changes. Furthermore, transgene-mediated inhibition of TRPC channels in mice dramatically reduced calcium influx and dystrophic disease manifestations associated with the mdx mutation (dystrophin gene) and deletion of the δ-sarcoglycan (Scgd) gene. These results demonstrate that calcium itself is sufficient to induce muscular dystrophy in vivo, and that TRPC channels are key disease initiators downstream of the unstable membrane that characterizes many types of muscular dystrophy.

Extracellular Signal-Regulated Kinases 1 and 2 Regulate the Balance Between Eccentric and Concentric Cardiac Growth

Rationale: An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, whereas volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood. Objective: To determine the role of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in regulating the cardiac hypertrophic response. Methods and Results: Here, we used mice lacking all ERK1/2 protein in the heart (Erk1−/− Erk2fl/fl-Cre) and mice expressing activated mitogen-activated protein kinase kinase (Mek)1 in the heart to induce ERK1/2 signaling, as well as mechanistic experiments in cultured myocytes to assess cellular growth characteristics associated with this signaling pathway. Although genetic deletion of all ERK1/2 from the mouse heart did not block the cardiac hypertrophic response per se, meaning that the heart still increased in weight with both aging and pathological stress stimulation, it did dramatically alter how the heart grew. For example, adult myocytes from hearts of Erk1−/− Erk2fl/fl-Cre mice showed preferential eccentric growth (lengthening), whereas myocytes from Mek1 transgenic hearts showed concentric growth (width increase). Isolated adult myocytes acutely inhibited for ERK1/2 signaling by adenoviral gene transfer showed spontaneous lengthening, whereas infection with an activated Mek1 adenovirus promoted constitutive ERK1/2 signaling and increased myocyte thickness. A similar effect was observed in engineered heart tissue under cyclic stretching, where ERK1/2 inhibition led to preferential lengthening. Conclusions: Taken together, these data demonstrate that the ERK1/2 signaling pathway uniquely regulates the balance between eccentric and concentric growth of the heart.Rationale An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, while volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood.

The IP3 Receptor Regulates Cardiac Hypertrophy in Response to Select Stimuli

Rationale: Inositol 1,4,5-trisphosphate (IP3) is a second messenger that regulates intracellular Ca2+ release through IP3 receptors located in the sarco(endo)plasmic reticulum of cardiac myocytes. Many prohypertrophic G protein–coupled receptor (GPCR) signaling events lead to IP3 liberation, although its importance in transducing the hypertrophic response has not been established in vivo. Objective: Here, we generated conditional, heart-specific transgenic mice with both gain- and loss-of-function for IP3 receptor signaling to examine its hypertrophic growth effects following pathological and physiological stimulation. Methods and Results: Overexpression of the mouse type-2 IP3 receptor (IP3R2) in the heart generated mild baseline cardiac hypertrophy at 3 months of age. Isolated myocytes from overexpressing lines showed increased Ca2+ transients and arrhythmias in response to endothelin-1 stimulation. Although low levels of IP3R2 overexpression failed to augment/synergize cardiac hypertrophy following 2 weeks of pressure-overload stimulation, such levels did enhance hypertrophy following 2 weeks of isoproterenol infusion, in response to G&agr;q overexpression, and/or in response to exercise stimulation. To inhibit IP3 signaling in vivo, we generated transgenic mice expressing an IP3 chelating protein (IP3-sponge). IP3-sponge transgenic mice abrogated cardiac hypertrophy in response to isoproterenol and angiotensin II infusion but not pressure-overload stimulation. Mechanistically, IP3R2-enhanced cardiac hypertrophy following isoproterenol infusion was significantly reduced in the calcineurin-A&bgr;–null background. Conclusion: These results indicate that IP3-mediated Ca2+ release plays a central role in regulating cardiac hypertrophy downstream of GPCR signaling, in part, through a calcineurin-dependent mechanism.

ERK1/2 regulate the balance between eccentric and concentric cardiac growth

Rationale: An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, whereas volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood. Objective: To determine the role of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in regulating the cardiac hypertrophic response. Methods and Results: Here, we used mice lacking all ERK1/2 protein in the heart (Erk1−/− Erk2fl/fl-Cre) and mice expressing activated mitogen-activated protein kinase kinase (Mek)1 in the heart to induce ERK1/2 signaling, as well as mechanistic experiments in cultured myocytes to assess cellular growth characteristics associated with this signaling pathway. Although genetic deletion of all ERK1/2 from the mouse heart did not block the cardiac hypertrophic response per se, meaning that the heart still increased in weight with both aging and pathological stress stimulation, it did dramatically alter how the heart grew. For example, adult myocytes from hearts of Erk1−/− Erk2fl/fl-Cre mice showed preferential eccentric growth (lengthening), whereas myocytes from Mek1 transgenic hearts showed concentric growth (width increase). Isolated adult myocytes acutely inhibited for ERK1/2 signaling by adenoviral gene transfer showed spontaneous lengthening, whereas infection with an activated Mek1 adenovirus promoted constitutive ERK1/2 signaling and increased myocyte thickness. A similar effect was observed in engineered heart tissue under cyclic stretching, where ERK1/2 inhibition led to preferential lengthening. Conclusions: Taken together, these data demonstrate that the ERK1/2 signaling pathway uniquely regulates the balance between eccentric and concentric growth of the heart.Rationale An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, while volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood.

A Thrombospondin-Dependent Pathway for a Protective ER Stress Response

Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a function for Thbs as ER-resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury, whereas Thbs4(-/-) mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. Thbs bind the ER lumenal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4(-/-) mice showed blunted activation of Atf6α and other ER stress-response factors with injury, and Thbs4-mediated protection was lost upon Atf6α deletion. Hence, Thbs can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

DUSP6 (MKP3) Null Mice Show Enhanced ERK1/2 Phosphorylation at Baseline and Increased Myocyte Proliferation in the Heart Affecting Disease Susceptibility

The strength and duration of mitogen-activated protein kinase signaling is regulated through phosphorylation and dephosphorylation by dedicated dual-specificity kinases and phosphatases, respectively. Here we investigated the physiological role that extracellular signal-regulated kinases 1/2 (ERK1/2) dephosphorylation plays in vivo through targeted disruption of the gene encoding dual-specificity phosphatase 6 (Dusp6) in the mouse. Dusp6-/- mice, which were viable, fertile, and otherwise overtly normal, showed an increase in basal ERK1/2 phosphorylation in the heart, spleen, kidney, brain, and fibroblasts, but no change in ERK5, p38, or c-Jun N-terminal kinases activation. However, loss of Dusp6 did not increase or prolong ERK1/2 activation after stimulation, suggesting that its function is more dedicated to basal ERK1/2 signaling tone. In-depth analysis of the physiological effect associated with increased baseline ERK1/2 signaling was performed in cultured mouse embryonic fibroblasts (MEFs) and the heart. Interestingly, mice lacking Dusp6 had larger hearts at every age examined, which was associated with greater rates of myocyte proliferation during embryonic development and in the early postnatal period, resulting in cardiac hypercellularity. This increase in myocyte content in the heart was protective against decompensation and hypertrophic cardiomyopathy following long term pressure overload and myocardial infarction injury in adult mice. Dusp6-/- MEFs also showed reduced apoptosis rates compared with wild-type MEFs. These results demonstrate that ERK1/2 signaling is physiologically restrained by DUSP6 in coordinating cellular development and survival characteristics, directly impacting disease-responsiveness in adulthood.