Allen J. York

Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart

The transcription factor GATA4 is a critical regulator of cardiac gene expression, modulating cardiomyocyte differentiation and adaptive responses of the adult heart. We report what we believe to be a novel function for GATA4 in murine cardiomyocytes as a nodal regulator of cardiac angiogenesis. Conditional overexpression of GATA4 within adult cardiomyocytes increased myocardial capillary and small conducting vessel densities and increased coronary flow reserve and perfusion-dependent cardiac contractility. Coculture of HUVECs with either GATA4-expressing cardiomyocytes or with myocytes expressing a dominant-negative form of GATA4 enhanced or reduced HUVEC tube formation, respectively. Expression of GATA4 in skeletal muscle by adenoviral gene transfer enhanced capillary densities and hindlimb perfusion following femoral artery ablation. Deletion of Gata4 specifically from cardiomyocytes reduced myocardial capillary density and prevented pressure overload-augmented angiogenesis in vivo. GATA4 induced the angiogenic factor VEGF-A, directly binding the Vegf-A promoter and enhancing transcription. GATA4-overexpressing mice showed increased levels of cardiac VEGF-A, while Gata4-deleted mice demonstrated decreased VEGF-A levels. The induction of HUVEC tube formation in GATA4-overexpressing cocultured myocytes was blocked with a VEGF receptor antagonist. Pressure overload-induced dysfunction in Gata4-deleted hearts was partially rescued by adenoviral gene delivery of VEGF and angiopoietin-1. To our knowledge, these results demonstrate [corrected] a previously unrecognized function for GATA4 as a regulator of cardiac angiogenesis through a nonhypoxic, load, and/or disease-responsive mechanism.

Genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo

MAPK signaling pathways function as critical regulators of cellular differentiation, proliferation, stress responsiveness, and apoptosis. One branch of the MAPK signaling pathway that culminates in ERK1/2 activation is hypothesized to regulate the growth and adaptation of the heart to both physiologic and pathologic stimuli, given its known activation in response to virtually every stress- and agonist-induced hypertrophic stimulus examined to date. Here we investigated the requirement of ERK1/2 signaling in mediating the cardiac hypertrophic growth response in Erk1−/− and Erk2+/− mice, as well as in transgenic mice with inducible expression of an ERK1/2-inactivating phosphatase in the heart, dual-specificity phosphatase 6. Although inducible expression of dual-specificity phosphatase 6 in the heart eliminated ERK1/2 phosphorylation at baseline and after stimulation without affecting any other MAPK, it did not diminish the hypertrophic response to pressure overload stimulation, neuroendocrine agonist infusion, or exercise. Similarly, Erk1−/− and Erk2+/− mice showed no reduction in pathologic or physiologic stimulus-induced cardiac growth in vivo. However, blockade or deletion of cardiac ERK1/2 did predispose the heart to decompensation and failure after long-term pressure overload in conjunction with an increase in myocyte TUNEL. Thus, ERK1/2 signaling is not required for mediating physiologic or pathologic cardiac hypertrophy in vivo, although it does play a protective role in response to pathologic stimuli.

Genetic Deletion of Myostatin From the Heart Prevents Skeletal Muscle Atrophy in Heart Failure

Background— Cardiac cachexia is characterized by an exaggerated loss of skeletal muscle, weakness, and exercise intolerance, although the cause of these effects remains unknown. Here, we hypothesized that the heart functions as an endocrine organ in promoting systemic cachexia by secreting peptide factors such as myostatin. Myostatin is a cytokine of the transforming growth factor-&bgr; superfamily that is known to control muscle wasting. Methods and Results— We used a Cre/loxP system to ablate myostatin (Mstn gene) expression in a cell type–specific manner. As expected, elimination of Mstn selectively in skeletal muscle with a myosin light chain 1f (MLC1f)-cre allele induced robust hypertrophy in all skeletal muscle. However, heart-specific deletion of Mstn with an Nkx2.5-cre allele did not alter baseline heart size or secondarily affect skeletal muscle size, but the characteristic wasting and atrophy of skeletal muscle that typify heart failure were not observed in these heart-specific null mice, indicating that myocardial myostatin expression controls muscle atrophy in heart failure. Indeed, myostatin levels in the plasma were significantly increased in wild-type mice subjected to pressure overload–induced cardiac hypertrophy but not in Mstn heart-specific deleted mice. Moreover, cardiac-specific overexpression of myostatin, which increased circulating levels of myostatin by 3- to 4-fold, caused a reduction in weight of the quadriceps, gastrocnemius, soleus, and even the heart itself. Finally, to investigate myostatin as a potential therapeutic target for the treatment of muscle wasting in heart failure, we infused a myostatin blocking antibody (JA-16), which promoted greater maintenance of muscle mass in heart failure. Conclusions— Myostatin released from cardiomyocytes induces skeletal muscle wasting in heart failure. Targeted inhibition of myostatin in cardiac cachexia might be a therapeutic option in the future.

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.

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.

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.

Decreased cardiac L-type Ca 2+channel activity induces hypertrophy and heart failure in mice

Antagonists of L-type Ca²⁺ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C⁻/⁺ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C⁻/⁺ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C⁻/⁺ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C⁺/⁺ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca²⁺ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease.

Plasma membrane Ca2+-ATPase isoform 4 antagonizes cardiac hypertrophy in association with calcineurin inhibition in rodents

How Ca2+-dependent signaling effectors are regulated in cardiomyocytes, given the extreme cytoplasmic Ca2+ concentration changes that underlie contraction, remains unknown. Cardiomyocyte plasma membrane Ca2+-ATPase (PMCA) extrudes Ca2+ but has little effect on excitation-contraction coupling, suggesting its potential role in controlling Ca2+-dependent signaling effectors such as calcineurin. We generated cardiac-specific inducible PMCA4b transgenic mice that displayed normal global Ca2+ transient and cellular contraction levels and reduced cardiac hypertrophy following transverse aortic constriction (TAC) or phenylephrine/Ang II infusion, but showed no reduction in exercise-induced hypertrophy. Transgenic mice were protected from decompensation and fibrosis following long-term TAC. The PMCA4b transgene reduced the hypertrophic augmentation associated with transient receptor potential canonical 3 channel overexpression, but not that associated with activated calcineurin. Furthermore, Pmca4 gene-targeted mice showed increased cardiac hypertrophy and heart failure events after TAC. Physical associations between PMCA4b and calcineurin were enhanced by TAC and by agonist stimulation of cultured neonatal cardiomyocytes. PMCA4b reduced calcineurin nuclear factor of activated T cell-luciferase activity after TAC and in cultured neonatal cardiomyocytes after agonist stimulation. PMCA4b overexpression inhibited cultured cardiomyocyte hypertrophy following agonist stimulation, but much less so in a Ca2+ pumping-deficient PMCA4b mutant. Thus, Pmca4b likely reduces the local Ca2+ signals involved in reactive cardiomyocyte hypertrophy via calcineurin regulation.

The Transcription Factor GATA-6 Regulates Pathological Cardiac Hypertrophy

Rationale: The transcriptional code that programs maladaptive cardiac hypertrophy involves the zinc finger–containing DNA binding factor GATA-4. The highly related transcription factor GATA-6 is also expressed in the adult heart, although its role in controlling the hypertrophic program is unknown. Objective: To determine the role of GATA-6 in cardiac hypertrophy and homeostasis. Methods and Results: Here, we performed a cardiomyocyte-specific conditional gene targeting approach for Gata6, as well as a transgenic approach to overexpress GATA-6 in the mouse heart. Deletion of Gata6-loxP with Nkx2.5-cre produced late embryonic lethality with heart defects, whereas deletion with &bgr;-myosin heavy chain-cre (&bgr;MHC-cre) produced viable adults with >95% loss of GATA-6 protein in the heart. These latter mice were subjected to pressure overload–induced hypertrophy for 2 and 6 weeks, which showed a significant reduction in cardiac hypertrophy similar to that observed Gata4 heart-specific deleted mice. Gata6-deleted mice subjected to pressure overload also developed heart failure, whereas control mice maintained proper cardiac function. Gata6-deleted mice also developed less cardiac hypertrophy following 2 weeks of angiotensin II/phenylephrine infusion. Controlled GATA-6 overexpression in the heart induced hypertrophy with aging and predisposed to greater hypertrophy with pressure overload stimulation. Combinatorial deletion of Gata4 and Gata6 from the adult heart resulted in dilated cardiomyopathy and lethality by 16 weeks of age. Mechanistically, deletion of Gata6 from the heart resulted in fundamental changes in the levels of key regulatory genes and myocyte differentiation–specific genes. Conclusions: These results indicate that GATA-6 is both necessary and sufficient for regulating the cardiac hypertrophic response and differentiated gene expression, both alone and in coordination with GATA-4.

Cdc42 is an antihypertrophic molecular switch in the mouse heart.

To improve contractile function, the myocardium undergoes hypertrophic growth without myocyte proliferation in response to both pathologic and physiologic stimulation. Various membrane-bound receptors and intermediate signal transduction pathways regulate the induction of cardiac hypertrophy, but the cardioprotective regulatory pathways or effectors that antagonize cardiac hypertrophy remain poorly understood. Here we identify the small GTPase Cdc42 as a signaling intermediate that restrained the cardiac growth response to physiologic and pathologic stimuli. Cdc42 was specifically activated in the heart after pressure overload and in cultured cardiomyocytes by multiple agonists. Mice with a heart-specific deletion of Cdc42 developed greater cardiac hypertrophy at 2 and 8 weeks of stimulation and transitioned more quickly into heart failure than did wild-type controls. These mice also displayed greater cardiac hypertrophy in response to neuroendocrine agonist infusion for 2 weeks and, more remarkably, enhanced exercise-induced hypertrophy and sudden death. These pathologies were associated with an inability to activate JNK following stimulation through a MEKK1/MKK4/MKK7 pathway, resulting in greater cardiac nuclear factor of activated T cells (NFAT) activity. Restoration of cardiac JNK signaling with an Mkk7 heart-specific transgene reversed the enhanced growth effect. These results identify what we believe to be a novel antihypertrophic and protective cardiac signaling pathway, whereby Cdc42-dependent JNK activation antagonizes calcineurin-NFAT activity to reduce hypertrophy and prevent transition to heart failure.