Federica Accornero

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.

Cardiac Overexpression of Melusin Protects From Dilated Cardiomyopathy Due to Long-Standing Pressure Overload

We have previously shown that genetic ablation of melusin, a muscle specific &bgr; 1 integrin interacting protein, accelerates left ventricle (LV) dilation and heart failure in response to pressure overload. Here we show that melusin expression was increased during compensated cardiac hypertrophy in mice subjected to 1 week pressure overload, but returned to basal levels in LV that have undergone dilation after 12 weeks of pressure overload. To better understand the role of melusin in cardiac remodeling, we overexpressed melusin in heart of transgenic mice. Echocardiography analysis indicated that melusin over-expression induced a mild cardiac hypertrophy in basal conditions (30% increase in interventricular septum thickness) with no obvious structural and functional alterations. After prolonged pressure overload (12 weeks), melusin overexpressing hearts underwent further hypertrophy retaining concentric LV remodeling and full contractile function, whereas wild-type LV showed pronounced chamber dilation with an impaired contractility. Analysis of signaling pathways indicated that melusin overexpression induced increased basal phosphorylation of GSK3&bgr; and ERK1/2. Moreover, AKT, GSK3&bgr; and ERK1/2 were hyper-phosphorylated on pressure overload in melusin overexpressing compared with wild-type mice. In addition, after 12 weeks of pressure overload LV of melusin overexpressing mice showed a very low level of cardiomyocyte apoptosis and stromal tissue deposition, as well as increased capillary density compared with wild-type. These results demonstrate that melusin overexpression allows prolonged concentric compensatory hypertrophy and protects against the transition toward cardiac dilation and failure in response to long-standing pressure overload.

Placental Growth Factor Regulates Cardiac Adaptation and Hypertrophy Through a Paracrine Mechanism

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

Modulation of chromatin position and gene expression by HDAC4 interaction with nucleoporins.

The histone deacetylase HDAC4 associates with the nuclear pore complex component Nup155 to modulate gene expression during cardiomyocyte hypertrophy.The nuclear lamina is increasingly being appreciated for its epigenetic role in regulating gene expression. The nuclear lamina underlies the inner nuclear membrane and, in post mitotic cells, is composed of a latticework primarily formed by the intermediate filament protein, lamin A/C. Although not well defined, lamin-associated domains have been described, and these domains are determined by DNA sequence and chromatin conformation. Lamin-associated domains are positioned to mediate the interaction with the nuclear membrane, where they contribute to transcriptional regulation. Although lamin-associated domains are primarily considered to be repressive in nature, those nearer to nuclear pores may actually promote transcription. Mutations in LMNA, the gene encoding lamins A and C, are a relatively common cause of inherited cardiomyopathy. As substantial data supports a role for the lamina in its interaction with chromatin and gene regulation, we examined the role of a genetically disrupted lamina and the consequences thereof. A dominant LMNA mutation, E161K, that causes inherited cardiomyopathy was studied. Gene expression changes were profiled in a human cardiomyopathic E161K heart, and it was found that chromosome 13 had a high percentage of misexpressed genes. Chromosome 13 was also found to be less tightly associated with the nuclear membrane in E161K mutant cells, thereby linking abnormal gene expression and intranuclear position. These and other studies support a role for the nuclear membrane as an active regulator of gene expression and provide additional support that disrupting this regulation is a mechanism of human disease.

Unrestrained p38 MAPK Activation in Dusp1/4 Double-Null Mice Induces Cardiomyopathy

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

The mammalian CHORD-containing protein melusin is a stress response protein interacting with Hsp90 and Sgt1

MINT‐6538515:melusin (Q9R000)physically interacts (MI:0218) with Hsp90 (P07901) by anti bait co-immunoprecipitation (MI:0006) MINT‐6538566, MINT‐6538556:Hsp90 (P07901) physically interacts (MI:0218) with melusin (Q9R000) by cross-linking studies (MI:0030) MINT‐6538524:melusin (Q9R000) physically interacts (MI:0218) with Sgt1 (Q9CS74) by anti tag co-immunoprecipitation (MI:0007) MINT‐6538595:Hsp90 (P07901) physically interacts (MI:0218) with melusin (Q9R000) by enzyme linked immunosorbent assay (MI:0411) MINT‐6538543:melusin (Q9R000) physically interacts (MI:0218) with Sgt1 (Q9CS74) by anti bait co-immunoprecipitation (MI:0006) MINT‐6538580: melusin (Q9R000) physically interacts (MI:0218) with Hsp90 (P07901) by surface plasmon resonance (MI:0107)

STIM1 elevation in the heart results in aberrant Ca2 + handling and cardiomyopathy

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

Genetic Analysis of Connective Tissue Growth Factor as an Effector of Transforming Growth Factor β Signaling and Cardiac Remodeling

ABSTRACT The matricellular secreted protein connective tissue growth factor (CTGF) is upregulated in response to cardiac injury or with transforming growth factor β (TGF-β) stimulation, where it has been suggested to function as a fibrotic effector. Here we generated transgenic mice with inducible heart-specific CTGF overexpression, mice with heart-specific expression of an activated TGF-β mutant protein, mice with heart-specific deletion of Ctgf, and mice in which Ctgf was also deleted from fibroblasts in the heart. Remarkably, neither gain nor loss of CTGF in the heart affected cardiac pathology and propensity toward early lethality due to TGF-β overactivation in the heart. Also, neither heart-specific Ctgf deletion nor CTGF overexpression altered cardiac remodeling and function with aging or after multiple acute stress stimuli. Cardiac fibrosis was also unchanged by modulation of CTGF levels in the heart with aging, pressure overload, agonist infusion, or TGF-β overexpression. However, CTGF mildly altered the overall cardiac response to TGF-β when pressure overload stimulation was applied. CTGF has been proposed to function as a critical TGF-β effector in underlying tissue remodeling and fibrosis throughout the body, although our results suggest that CTGF is of minimal importance and is an unlikely therapeutic vantage point for the heart.

Enhanced Ca2+ influx from STIM1–Orai1 induces muscle pathology in mouse models of muscular dystrophy

Muscular dystrophy is a progressive muscle wasting disease that is thought to be initiated by unregulated Ca(2+) influx into myofibers leading to their death. Store-operated Ca(2+) entry (SOCE) through sarcolemmal Ca(2+) selective Orai1 channels in complex with STIM1 in the sarcoplasmic reticulum is one such potential disease mechanism for pathologic Ca(2+) entry. Here, we generated a mouse model of STIM1 overexpression in skeletal muscle to determine whether this type of Ca(2+) entry could induce muscular dystrophy. Myofibers from muscle-specific STIM1 transgenic mice showed a significant increase in SOCE in skeletal muscle, modeling an observed increase in the same current in dystrophic myofibers. Histological and biochemical analysis of STIM1 transgenic mice showed fulminant muscle disease characterized by myofiber necrosis, swollen mitochondria, infiltration of inflammatory cells, enhanced interstitial fibrosis and elevated serum creatine kinase levels. This dystrophic-like disease in STIM1 transgenic mice was abrogated by crossing in a transgene expressing a dominant-negative Orai1 (dnOrai1) mutant. The dnOrai1 transgene also significantly reduced the severity of muscular dystrophy in both mdx (dystrophin mutant mice) and δ-sarcoglycan-deficient (Sgcd(-/-)) mouse models of disease. Hence, Ca(2+) influx across an unstable sarcolemma due to increased activity of a STIM1-Orai1 complex is a disease determinant in muscular dystrophy, and hence, SOCE represents a potential therapeutic target.