Beta-myosin Heavy Chain Expression Research Articles

Sustained Elevated Circulating Activin A Impairs Global Longitudinal Strain in Pregnant Rats: A Potential Mechanism for Preeclampsia-Related Cardiac Dysfunction.

Mediators of cardiac injury in preeclampsia are not well understood. Preeclamptic women have decreased cardiac global longitudinal strain (GLS), a sensitive measure of systolic function that indicates fibrosis and tissue injury. GLS is worse in preeclampsia compared to gestational hypertension, despite comparable blood pressure, suggesting that placental factors may be involved. We previously showed that Activin A, a pro-fibrotic factor produced in excess by the placenta in preeclampsia, predicts impaired GLS postpartum. Here, we hypothesized that chronic excess levels of Activin A during pregnancy induces cardiac dysfunction. Rats were assigned to sham or activin A infusion (1.25–6 µg/day) on a gestational day (GD) 14 (n = 6–10/group). All animals underwent blood pressure measurement and comprehensive echocardiography followed by euthanasia and the collection of tissue samples on GD 19. Increased circulating activin A (sham: 0.59 ± 0.05 ng/mL, 6 µg/day: 2.8 ± 0.41 ng/mL, p < 0.01) was associated with impaired GLS (Sham: −22.1 ± 0.8%, 6 µg/day: −14.7 ± 1.14%, p < 0.01). Activin A infusion (6 µg/day) increased beta-myosin heavy chain expression in heart tissue, indicating cardiac injury. In summary, our findings indicate that increasing levels of activin A during pregnancy induces cardiac dysfunction and supports the concept that activin A may serve as a possible mediator of PE-induced cardiac dysfunction.

Cardiac Thyrotropin-releasing Hormone Inhibition Improves Ventricular Function and Reduces Hypertrophy and Fibrosis After Myocardial Infarction in Rats

BackgroundCardiac thyrotropin-releasing hormone (TRH) is a tripeptide with still unknown functions. We demonstrated that the left ventricle (LV) TRH system is hyperactivated in spontaneously hypertensive rats and its inhibition prevented cardiac hypertrophy and fibrosis. Therefore, we evaluated whether in vivo cardiac TRH inhibition could improve myocardial function and attenuate ventricular remodeling in a rat model of myocardial infarction (MI). Methods and ResultsIn Wistar rats, MI was induced by a permanent left anterior descending coronary artery ligation. A coronary injection of a specific small interfering RNA against TRH was applied simultaneously. The control group received a scrambled small interfering RNA. Cardiac remodeling variables were evaluated one week later. In MI rats, TRH inhibition decreased LV end-diastolic (1.049 ± 0.102 mL vs 1.339 ± 0.102 mL, P < .05), and end-systolic volumes (0.282 ± 0.043 mL vs 0.515 ± 0.037 mL, P < .001), and increased LV ejection fraction (71.89 ± 2.80% vs 65.69 ± 2.85%, P < .05). Although both MI groups presented similar infarct size, small interfering RNA against TRH treatment attenuated the cardiac hypertrophy index and myocardial interstitial collagen deposition in the peri-infarct myocardium. These effects were accompanied by attenuation in the rise of transforming growth factor-β, collagen I, and collagen III, as well as the fetal genes (atrial natriuretic peptide, B-type natriuretic peptide, and beta myosin heavy chain) expression in the peri-infarct region. In addition, the expression of Hif1α and vascular endothelial growth factor significantly increased compared with all groups. ConclusionsCardiac TRH inhibition improves LV systolic function and attenuates ventricular remodeling after MI. These novel findings support the idea that TRH inhibition may serve as a new therapeutic strategy against the progression of heart failure.

Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease; however, its etiology remains largely unknown. We previously demonstrated that genetic variants in the MYH6 gene are significantly associated with HLHS. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from an HLHS-affected family trio (affected parent, unaffected parent, affected proband) carrying an MYH6-R443P head domain variant demonstrated dysmorphic sarcomere structure and increased compensatory MYH7 expression. Analysis of iPSC-CMs derived from the HLHS trio revealed that only beta myosin heavy chain expression was observed in CMs carrying the MYH6-R443P variant after differentiation day 15 (D15). Functional assessments performed between D20-D23 revealed that MYH6-R443P variant CMs contracted more slowly (40 ± 2 vs. 47 ± 2 contractions/min, P < 0.05), shortened less (5.6 ± 0.5 vs. 8.1 ± 0.7% of cell length, P < 0.05), and exhibited slower shortening rates (19.9 ± 1.7 vs. 28.1 ± 2.5 μm/s, P < 0.05) and relaxation rates (11.0 ± 0.9 vs. 19.7 ± 2.0 μm/s, P < 0.05). Treatment with isoproterenol had no effect on iPSC-CM mechanics. Using CRISPR/Cas9 gene editing technology, introduction of the R443P variant into the unaffected parent’s iPSCs recapitulated the phenotype of the proband’s iPSC-CMs, and conversely, correction of the R443P variant in the proband’s iPSCs rescued the cardiomyogenic differentiation, sarcomere organization, slower contraction (P < 0.05) and decreased velocity phenotypes (P < 0.0001). This is the first report to identify that cardiac tissues from HLHS patients with MYH6 variants can exhibit sarcomere disorganization in atrial but not ventricular tissues. This new discovery was not unexpected, since MYH6 is expressed predominantly in the postnatal atria in humans. These findings demonstrate the feasibility of employing patient-derived iPSC-CMs, in combination with patient cardiac tissues, to gain mechanistic insight into how genetic variants can lead to HLHS. Results from this study suggest that decreased contractility of CMs due to sarcomere disorganization in the atria may effect hemodynamic changes preventing development of a normal left ventricle.

Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5′-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells

Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5′-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy.

Right ventricular plasticity in a porcine model of chronic pressure overload

Ventricular-arterial coupling is a measure of the relationship between ventricular contractility and afterload. We sought to determine the relationship between ventricular-arterial coupling and right ventricular (RV) remodeling in a novel porcine model of progressive pulmonary hypertension (PH). Chronic PH was induced in pigs by ligation of the left pulmonary artery (PA) followed by 5 weekly injections of cyanoacrylate to progressively obstruct the right lower lobe arteries (PH group, n = 10). At 6 weeks, 5 PH animals underwent reperfusion of the left lung through conduit anastomosis to decrease RV afterload, whereas 5 other animals received no treatment. Five sham-operated piglets were used as controls. RV function was assessed using echocardiography and conductance catheterization. RV gene expression of beta-myosin heavy chain (β-MHC) and B-type natriuretic peptide (BNP) were quantified by polymerase chain reaction. At 6 weeks, compared with controls, the PH group had higher mean PA pressure (32 ± 6 vs 14 ± 2 mm Hg, p < 0.01). The increase in RV elastance was insufficient to compensate for the increase in pulmonary arterial elastance in the PH group and altered ventricular-arterial coupling occurred (0.65 ± 0.16 vs 1.28 ± 0.14, p < 0.01). The degree of ventricular-arterial uncoupling was related to RV enlargement and systolic dysfunction. Ventricular-arterial uncoupling and increased RV mass index were associated with up-regulation of β-MHC and BNP expression. Ventricular-arterial coupling is closely associated with ventricular remodeling and systolic function as well as contractile and BNP gene expression. Dynamic changes in myosin expression may determine RV work efficiency in PH.

The effect of Intermedin on Angiotensin II and Endothelin-1 Induced Ventricular Myocyte Hypertrophy in Neonatal Rat

Intermedin (IMD), a novel peptide related to calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), may have localized actions as a modulator of cardiac function. The aim of the study is to explore the effect of IMD on angiotensin II (Ang II) and endothelin-1 (ET-1) induced hypertrophy in ventricular myocytes of neonatal rat and to try to elucidate the possible mechanism. Neonatal rat cardiomyocytes were cultured in serum-free medium with and without AngII (1 micromol/L) or ET-1 (60 micromol/L) in the presence and absence of IMD (1 micromol/L). Hypertrophic responses (including cell surface area, alpha-actin, and beta-myosin heavy chain mRNA expression) and cardiomyocyte expression of NADPH oxidase gp91phox were determined. Ang II induced increases in cardiomyocyte size to 305 +/- 32 microm2 (n = 198, p < 0.05, at 48 hours), alpha-actin expression to 4 +/- 2.8-fold (n = 6, p < 0.05, at 48 hours) and beta-myosin heavy chain expression to 11 +/- 4.8-fold (n = 6, p < 0.05, at 48 hours), and expression of the gp91phox subunit of NADPH oxidase to 29.4 +/- 12.7-fold (n = 6, p < 0.05, at 48 hours). These effects were all significantly inhibited by IMD; cardiomyocyte size, alpha-actin expression, beta-myosin heavy chain expression, and gp91phox expression were reduced to 265 +/- 32 microm2 (n = 374, p < 0.05), 3.0 +/- 1.7-fold (n = 6, p < 0.05), 8.7 +/- 4.9-fold (n = 6, p < 0.05), 3.9 +/- 3-fold (n = 6, p < 0.05), respectively. IMD also significantly inhibited ET1-induced increases in cardiomyocyte size and superoxide generation. IMD exerts an antihypertrophic effect on neonatal cardiomyocytes by reduced levels of superoxide, suggesting that an antioxidant action contributes to the antihypertrophic actions of IMD.

Hypothyroidism Effects on Wild Type and Mutant Rats with Altered Cardiac Titin Expression

Thyroid hormone levels play an important role in cardiac regulation, and a state of hypothyroidism leads to dilated forms of cardiomypathy. Inappropriate thyroid levels can also impact the sarcomeric protein titin, which is responsible for maintaining passive tension and structural integrity. The effects of hypothyroidism were studied after administration of propylthiouracil (PTU) to wild type (Wt) and homozygous mutant (Hm) rats (Greaser et al J Mol Cellul Cardiol 44:983, 2008) (the latter of which express a giant titin isoform). Enzyme-linked immunosorbent assay results demonstrated that PTU lowered thyroxine levels in the treated rats. Electrophoretic cardiac myosin heavy chain analysis indicated that hypothyroidism induced a transition to predominantly beta myosin heavy chain expression, while Wt control samples continued to express an age-appropriate range of alpha and beta isoforms. PTU-treated Wt rats had a larger N2BA to N2B titin ratio, and the slower migrating N2BA was in higher proportion than the faster N2BA. PTU did not affect titin isoform expression in Hm. Because it is believed that thyroid hormone regulates titin isoform expression through the PI3K/Akt pathway, we investigated several participants by immunoblotting. The Akt PH domain was not altered by PTU administration, although expression was higher in Hm rats. Akt phospho-Ser473 did not vary between treatments, but mTOR phospho-Ser2448 demonstrated a higher phosphorylation state in Wt. Echocardiography data showed significantly decreased fractional shortening in Hm control, Hm PTU, and Wt PTU relative to Wt control. Wt control also exhibited a higher ejection fraction and a lower isovolumic relaxation time compared to the other treatments. It can be concluded that hypothyroidism has significant effects on titin isoform expression and other cardiac functional characteristics. Supported by NIH HL77196.

Ca2+/calmodulin‐dependent protein kinase IIδ orchestrates G‐protein‐coupled receptor and electric field stimulation‐induced cardiomyocyte hypertrophy

1. G-Protein-coupled receptors (GPCR) and electrical field stimulation (EFS) regulate cardiac function and pathological remodelling, including cardiac hypertrophy. Cardiac Ca(2+)/calmodulin-dependent protein kinase (CaMK) IIdelta expression and activity are altered in cardiac hypertrophy and heart failure. The aim of the present study was to determine the effects of CaMKIIdelta isoforms on neonatal rat cardiomyocyte hypertrophy induced by GPCR and EFS. 2. Cardiac hypertrophy was induced by angiotensin II, phenylephrine or EFS and was confirmed by increases in cell volume, [(3)H]-leucine incorporation, sarcomere assembly and mRNA expression of atrial natriuretic factor and beta-myosin heavy chain. The effects of the CaMKII inhibitors KN93 and autocamtide 2-related inhibitory peptide (AIP) on cardiomyocyte hypertrophy were investigated, as was the effect of overexpression of dominate negative CaMKIIdelta. 3. Cardiomyocyte hypertrophy was inhibited by the CaMKII inhibitors KN93 and AIP and by overexpression of dominate negative CaMKIIdelta, but was potentiated by overexpression of wild-type CaMKIIdeltaB or CaMKIIdeltaC. Activation of CaMKII by GPCR agonists or EFS was inhibited by the CaMKII inhibitors. 4. The GPCR agonists and EFS synergistically activated CaMKII and upregulated CaMKIIdeltaB and CaMKIIdeltaC mRNA expression and protein synthesis. All these effects were abolished by the CaMKII inhibitors. 5. The findings of the present study indicate that CaMKII orchestrates additive prohypertrophic factors between GPCR agonists and EFS. Thus, CaMKII may be a useful target in the clinical treatment of hypertrophy and cardiac remodelling.

Activation of AMPK inhibits cardiomyocyte hypertrophy by modulating of the FOXO1/MuRF1 signaling pathway in vitro

To examine the inhibitory effects of adenosine monophosphate-activated protein kinase (AMPK) activation on cardiac hypertrophy in vitro and to investigate the underlying molecular mechanisms. Cultured neonatal rat cardiomyocytes were treated with the specific AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the specific AMPK antagonist Compound C, and then stimulated with phenylephrine (PE). The Muscle RING finger 1 (MuRF1)-small interfering RNA (siRNA) was transfected into cardiomyocytes using Lipofectamine 2000. The surface area of cultured cardiomyocytes was measured using planimetry. The protein degradation was determined using high performance liquid chromatography (HPLC). The expression of beta-myosin heavy chain (beta-MHC) and MuRF1, as well as the phosphorylation levels of AMPK and Forkhead box O 1 (FOXO1), were separately measured using Western blot or real-time polymerase chain reaction. Activation of AMPK by AICAR 0.5 mmol/L inhibited PE-induced increase in cardiomyocyte area and beta-MHC protein expression and PE-induced decrease in protein degradation. Furthermore, AMPK activation increased the activity of transcription factor FOXO1 and up-regulated downstream atrogene MuRF1 mRNA and protein expression. Treatment of hypertrophied cardiomyocytes with Compound C 1 micromol/L blunted the effects of AMPK on cardiomyocyte hypertrophy and changes to the FOXO1/MuRF1 pathway. The effects of AICAR on cardiomyocyte hypertrophy were also blocked after MuRF1 was silenced by transfection of cardiomyocytes with MuRF1-siRNA. The present study demonstrates that AMPK activation attenuates cardiomyocyte hypertrophy by modulating the atrophy-related FOXO1/MuRF1 signaling pathway in vitro.

Scutellarin exerts its anti-hypertrophic effects via suppressing the Ca2+-mediated calcineurin and CaMKII signaling pathways

Scutellarin is a flavonoid extracted from a traditional Chinese herb, Erigeron breviscapus Hand Mazz, which has been broadly used in treating various cardiovascular diseases. In this study, we investigated its effect on cardiac hypertrophy and the underlying mechanism. Both in vitro and in vivo cardiac hypertrophy models were employed to explore the anti-hypertrophic action of scutellarin. We found that scutellarin significantly suppressed the hypertrophic growth of neonatal cardiac myocytes exposed to phenylephrine (PE) and mouse heart subjected to pressure overload induced by aortic banding, accompanied with the decreased expression of hypertrophic markers beta-myosin heavy chain and atrial natriuretic peptide. We then measured the change of free intracellular calcium using laser scanning confocal microscope. We found that scutellarin alleviated the increment of free intracellular calcium during cardiac hypertrophy either induced by PE or aortic banding. The expression of calcium downstream effectors calcineurin and phosphorylated calmodulin kinase II (CaMKII) were significantly suppressed by scutellarin. Our study indicated that scutellarin exerts its anti-hypertrophic activity via suppressing the Ca(2+)-mediated calcineurin and CaMKII pathways, which supports the observation that clinical application of scutellarin is beneficial for cardiovascular disease patients.

The Role of MEKK1 in Hypertrophic Cardiomyopathy

MEKK1 is a ubiquitously expressed mitogen activated protein kinase that is involved in tissue remodeling in a variety of settings including carotid artery blood flow cessation, wound healing, and breast adenocarcinoma intravasation. Here, we have tested the function of MEKK1 in genetic hypertrophic cardiomyopathy (HCM). MEKK1 was genetically deleted in C57Bl6/J mice expressing a mutant alpha-myosin heavy chain (HCM-MEKK1(-/-)). The absence of MEKK1 in HCM resulted in a more pronounced hypertrophy when compared to HCM mice with the MEKK1 gene intact without further increases in atrial natriuretic factor and beta-myosin heavy chain (MyHC) expression and fibrosis. Since MEKK1 is required for the induction of several tissue proteases, we tested the hypothesis that cardiac enlargement of HCM- MEKK1(-/-) mice was due to altered expression of urokinase-type plasminogen activator (uPA), JunB, matrix-metalloproteinase (MMP), and tissue inhibitors of MMPs (TIMPs). Because of its role in preventing apoptosis, we also tested the loss of MEKK1 on apoptotic mediators Bcl-2, cytochrome C, caspase-9, and caspase-3. uPA expression was decreased while JunB, MMP-9, caspase-9, and caspase-3 activities were elevated in HCM- MEKK1(-/-) hearts when compared to MEKK1(-/-), wild-type (WT), and HCM mice. Bcl-2 and Cyt C expression was elevated only in HCM mice. We conclude that the absence of MEKK1 induces a more pronounced cardiac hypertrophy to HCM through altered expression of proteases implicated in cardiac remodeling and increased apoptosis.

Systemic Trans-Splicing Adeno-Associated Viral Delivery Efficiently Transduces the Heart of Adult mdx Mouse, a Model for Duchenne Muscular Dystrophy

Trans-splicing adeno-associated viral (tsAAV) vectors hold great promise for delivering large therapeutic genes. One potential application is in the treatment of Duchenne muscular dystrophy (DMD). In this case, it is necessary to transduce whole body muscle. We demonstrated body-wide AAV-9 tsAAV transduction in normal neonatal mice. However, it was not clear whether such an approach would work in diseased mice. In this study we delivered the AAV-9 alkaline phosphatase (AP) tsAAV vector (3 x 10(12) vector genome particles per vector per mouse, tail vein injection) to 2-month-old mdx mice, the most widely used DMD model. Four months later, we observed widespread AP expression in the heart. It reached the same level as we have seen in normal neonatal puppy. Interestingly, myocardial transduction correlated with beta-myosin heavy chain expression but not with LamR, the putative AAV-9 receptor. AP expression was also detected in various skeletal muscles but at levels much lower than in normal newborn mice. Despite the existing inflammatory milieu, we did not see any appreciable increase in CD4(+) and CD8(+) T cells and macrophages in striated muscles after systemic tsAAV infection. In summary, our results have paved the way for tsAAV-mediated gene therapy for Duchenne cardiomyopathy.

A Family of microRNAs Encoded by Myosin Genes Governs Myosin Expression and Muscle Performance

Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.

Cardiac fibroblast paracrine factors alter impulse conduction and ion channel expression of neonatal rat cardiomyocytes

The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury results in fibrosis, which correlates with arrhythmia generation and heart failure. Here we systematically examined the effect of fibroblast-derived paracrine factors on electrical propagation in cardiomyocytes. Neonatal rat cardiac monolayers were exposed for 24 h to media conditioned by CFs. Optical mapping, sharp microelectrode recordings, quantitative RT-PCR, and immunostaining were used to assess the changes in the propagation and shape of the action potential and underlying changes in gene and protein expression. The fibroblast paracrine factors produced a 52% reduction in cardiac conduction velocity, a 217% prolongation of action potential duration, a 64% decrease of maximum capture rate, a 21% increase in membrane resting potential, and an 80% decrease of action potential upstroke velocity. These effects were dose dependent and partially reversible with removal of the conditioned media. No fibroblast proliferation, cardiomyocyte apoptosis, or decreased connexin-43 expression, phosphorylation, and function were found in conditioned cardiac cultures. In contrast, the expression of the fast sodium, inward rectifying potassium, and transient outward potassium channels were, respectively, reduced 3.8-, 6.6-fold, and to undetectable levels. The expression of beta-myosin heavy chain increased 17.4-fold. No electrophysiological changes were observed from media conditioned by CFs in the presence of cardiomyocytes. Paracrine factors from neonatal CFs alone produced significant electrophysiological changes in neonatal rat cardiomyocytes resembling those found in several cardiac pathologies.

The role of thyroid hormone nuclear receptors in the heart: evidence from pharmacological approaches

This review evaluates the hypothesis that the cardiac effects of amiodarone can be explained—at least partly—by the induction of a local ‘hypothyroid-like condition’ in the heart. Evidence supporting the hypothesis comprises the observation that amiodarone exerts an inhibitory effect on the binding of T3 to thyroid hormone receptors (TR) alpha-1 and beta-1 in vitro, and on the expression of particular T3-dependent genes in vivo. In the heart, amiodarone decreases heart rate and alpha myosin heavy chain expression (mediated via TR alpha-1), and increases sarcoplasmic reticulum calcium-activated ATPase and beta myosin heavy chain expression (mediated via TR beta-1). Recent data show a significant similarity in expression profiles of 8,435 genes in the heart of hypothyroid and amiodarone-treated animals, although similarities do not always exist in transcripts of ion channel genes. Induction of a hypothyroid cardiac phenotype by amiodarone may be advantageous by decreasing energy demands and increasing energy availability.

Inhibition of protein phosphatase 1 by inhibitor-2 exacerbates progression of cardiac failure in a model with pressure overload

The progression of human heart failure is associated with increased protein phosphatase 1 (PP1) activity, which leads to a higher dephosphorylation of cardiac regulatory proteins such as phospholamban. In this study, we tested the hypothesis whether the inhibitor-2 (I-2) of PP1 can mediate cardiac protection by inhibition of PP1 activity. We induced pressure overload by transverse aortic constriction (TAC) for 28 days in transgenic (TG) mice with heart-directed overexpression of a constitutively active form of I-2 (TG(TAC)) and wild-type littermates (WT(TAC)). Both groups were compared with sham-operated mice. TAC treatment resulted in comparable ventricular hypertrophy in both groups. However, TG(TAC) exhibited a higher atrial mass and an enhanced ventricular mRNA expression of beta-myosin heavy chain. The increased afterload was associated with the development of focal fibrosis in TG. Consistent with signs of overt heart failure, fractional shortening and diastolic function were impaired in TG(TAC) as revealed by Doppler echocardiography. The contractility was reduced in catheterized banded TG mice, which is in line with a depressed shortening of isolated myocytes. This is due to profoundly abnormal cytosolic Ca(2+) transients and a reduced stimulation of phosphorylation of phospholamban (PLB)(Ser16) after TAC in TG mice. Moreover, administration of isoproterenol was followed by a blunted contractile response in isolated myocytes of TG(TAC) mice. These results suggest that cardiac-specific overexpression of a constitutively active form of I-2 is deleterious for cardiac function under conditions of pressure overload. Thus, the long-term inhibition of PP1 by I-2 is not a therapeutic option in the treatment of heart failure.

Role of myosin heavy chain composition in the stretch activation response of rat myocardium

The speed and force of myocardial contraction during systolic ejection is largely dependent on the intrinsic contractile properties of cardiac myocytes. As the myosin heavy chain (MHC) isoform of cardiac muscle is an important determinant of the contractile properties of individual myocytes, we studied the effects of altered MHC isoform expression in rat myocardium on the mechanical properties of skinned ventricular preparations. Skinned myocardium from thyroidectomized rats expressing only the beta MHC isoform displayed rates of force redevelopment that were about 2.5-fold slower than in myocardium from hyperthyroid rats expressing only the alpha MHC isoform, but the amount of force generated at a given level of Ca2+ activation was not different. Because recent studies suggest that the stretch activation response in myocardium has an important role in systolic function, we also examined the effect of MHC isoform expression on the stretch activation response by applying a rapid stretch (1% of muscle length) to an otherwise isometrically contracting muscle fibre. Sudden stretch of myocardium resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force (i.e. stretch activation) to levels greater than prestretch force. beta MHC expression dramatically slowed the overall rate of the stretch activation response compared to expression of alpha MHC isoform; specifically, the rate of force decay was approximately 2-fold slower and the rate of delayed force development was approximately 2.5-fold slower. In contrast, MHC isoform had no effect on the amplitude of the stretch activation response. Collectively, these data show that expression of beta MHC in myocardium dramatically slows rates of cross-bridge recruitment and detachment which would be expected to decrease power output and contribute to depressed systolic function in end-stage heart failure.

REVERSING THE FETAL GENE PROGRAM IN THE SETTING OF PATHOLOGIC STIMULI IMPROVES CARDIOMYOCYTE FUNCTION.

Background Previously we found that abrogating activator protein 1 (AP-1) in neonatal rat ventricular myocytes subjected to pathologic stimuli resulted in reversal of the pathologic/fetal gene profile while maintaining agonist-induced myocyte hypertrophy. We now extend these observations to the adult rat cardiomyocyte (ARVM) and further investigate if reversing the gene program toward a physiologic profile in a pathologic model results in improved myocyte function. Methods and Results Hypertrophy was induced in cultured ARVMs using norepinephrine with propranolol pretreatment. Induction of the pathologic gene profile was observed, ie, expression of beta-myosin heavy chain (betaMHC), and atrial/brain natriuretic peptides (ANP/BNP) was increased, whereas expression for the alpha-myosin heavy chain (alphaMHC) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) genes was repressed. As with the neonatal cardiomyocyte experiments, the role of AP-1 in the hypertrophic phenotype was evaluated with the use of an adenoviral construct expressing a dominant negative mutant of the c-Fos proto-oncogene (AdAFos). Although AdAFos infection did not change the myocyte growth response, it resulted in the reversal of the gene profile to alpha-adrenergic stimulation, ie, up-regulation of alphaMHC/SERCA and repression of betaMHC and ANP/BNP. Translation of both MHC and SERCA proteins corresponded with their gene expression. Further, ARVM infected with Ad-AFos showed improved myocyte shortening, a surrogate marker for contractile function. Conclusion Abrogation of AP-1 transcription factor in the setting of alpha-adrenergic stimulation results in improved cardiomyocyte function.

TRPC6 fulfills a calcineurin signaling circuit during pathologic cardiac remodeling

The heart responds to injury and chronic pressure overload by pathologic growth and remodeling, which frequently result in heart failure and sudden death. Calcium-dependent signaling pathways promote cardiac growth and associated changes in gene expression in response to stress. The calcium/calmodulin-dependent phosphatase calcineurin, which signals to nuclear factor of activated T cells (NFAT) transcription factors, serves as a transducer of calcium signals and is sufficient and necessary for pathologic cardiac hypertrophy and remodeling. Transient receptor potential (TRP) proteins regulate cation entry into cells in response to a variety of signals, and in skeletal muscle, expression of TRP cation channel, subfamily C, member 3 (TRPC3) is increased in response to neurostimulation and calcineurin signaling. Here we show that TRPC6 was upregulated in mouse hearts in response to activated calcineurin and pressure overload, as well as in failing human hearts. Two conserved NFAT consensus sites in the promoter of the TRPC6 gene conferred responsiveness to cardiac stress. Cardiac-specific overexpression of TRPC6 in transgenic mice resulted in heightened sensitivity to stress, a propensity for lethal cardiac growth and heart failure, and an increase in NFAT-dependent expression of beta-myosin heavy chain, a sensitive marker for pathologic hypertrophy. These findings implicate TRPC6 as a positive regulator of calcineurin-NFAT signaling and a key component of a calcium-dependent regulatory loop that drives pathologic cardiac remodeling.

Antioxidant actions contribute to the antihypertrophic effects of atrial natriuretic peptide in neonatal rat cardiomyocytes

Reactive oxygen species (ROS) such as superoxide have been linked to the hypertrophic response of the heart to stimuli including angiotensin II (AngII), mechanical stretch, and pressure overload. We have previously demonstrated that cGMP and protein kinase G mediate the antihypertrophic actions of the natriuretic peptides in rat cardiomyocytes and isolated whole hearts. The impact of natriuretic peptides on cardiac ROS generation, however, has not been investigated. We tested the hypothesis that reduced superoxide accumulation contributes to the antihypertrophic action of atrial natriuretic peptide (ANP). Neonatal rat cardiomyocytes were cultured in serum-free medium with and without AngII (1 micromol/L) or endothelin-1 (ET(1), 60 nmol/L) in the presence and absence of ANP (1 micromol/L) or tempol (100 micromol/L). Hypertrophic responses, cardiomyocyte superoxide generation, and cardiomyocyte expression of NADPH oxidase were determined. AngII induced increases in cardiomyocyte size (to 176 +/- 9% n = 8 p < 0.001, at 48 h), beta-myosin heavy chain expression (to 4.0 +/- 1.6-fold n = 6 p < 0.05, at 48 h), c-fos expression (to 1.9 +/- 0.5-fold n = 7 p < 0.01, at 6 h), superoxide generation (to 181+/-21% n = 8 p < 0.005, at 24 h), and expression of the gp91phox subunit of NADPH oxidase (to 2.4 +/- 0.5-fold n = 7 p < 0.05, at 48 h). These effects were all significantly inhibited by ANP: cardiomyocyte size, beta-myosin heavy chain expression, c-fos expression, superoxide generation and gp91phox expression were reduced to 107 +/- 5% (n = 5 p < 0.05), 1.2 +/- 0.2-fold (n = 6 p < 0.05), 0.9 +/- 0.2-fold (n = 7 p < 0.05), 141 +/- 21% (n = 8 p < 0.05), and to 1.0 +/- 0.5-fold (n = 7 p < 0.05), respectively. These effects were mimicked by tempol. ANP and tempol also significantly inhibited ET1-induced increases in cardiomyocyte size and superoxide generation, but had no effect on markers of hypertrophy when studied alone. This data indicates that the antihypertrophic actions of ANP are accompanied by reduced levels of superoxide, suggesting an antioxidant action contributes to the antihypertrophic actions of ANP.