Myocyte Size Research Articles

Assessment of muscle mass and strength in mice.

Muscle weakness is an important phenotype of many diseases that is linked to impaired locomotion and increased mortality. The force that a muscle can generate is determined predominantly by muscle size, fiber type and the excitation-contraction coupling process. Here we describe methods for the histological assessment of whole muscle to determine fiber cross-sectional area and fiber type, determination of changes in myocyte size using C2C12 cells, in vivo functional tests and measurement of contractility in dissected whole muscles. The extensor digitorum longus and soleus muscles are ideally suited for whole-muscle contractility, and dissection of these muscles is described.

Abstract 4489: Angiotensin-(1-7) prevents fibrosis in doxorubicin-treated rats

Abstract Anthracyclines such as doxorubicin (Dox) are effective chemotherapeutic agents for multiple malignancies, including childhood leukemias and sarcomas. Despite the efficacy of these agents, anthracyclines can result in cumulative dose-dependent cardiac toxicity that manifests as cardiomyopathy with marked myocardial hypertrophy and fibrosis. This cardiotoxicity culminates in reduced ejection fraction, fractional shortening, and cardiac output, leading to congestive heart failure. Consequently, there is an unmet need for adjunct therapies to temper anthracycline-induced cardiotoxicity and enhance long-term quality of life in patients treated with anthracyclines, especially pediatric cancer patients. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous seven amino acid peptide hormone of the renin-angiotensin system with cardioprotective properties. Studies by us and others showed that Ang-(1-7) improved cardiac function as well as reduced cardiac hypertrophy and fibrosis in various animal models. In this study, we assessed whether adjunct Ang-(1-7) (24 μg/kg/hr) attenuates cardiotoxicity that results from acute (6 week) exposure of 5 week-old Sprague-Dawley rats to Dox, administered weekly at a cumulative dose of 15 mg/kg. Body weight was reduced in rats treated with Dox, in the presence or absence of Ang-(1-7); however, the heart weight/body weight ratio was increased in Dox-treated rats while co-administration with Ang-(1-7) prevented the Dox-mediated increase. Myocyte cross-sectional area was not changed by administration of Dox or Ang-(1-7). Dox administration caused significant increases in cardiac fibrosis (an increase in interstitial fibrosis from 0.9±0.1% to 1.5±0.2%, p<0.001 compared to sham, and perivascular fibrosis from 16.0±1.1% to 23.6±3.0%, p<0.05; n = 3-8), which was prevented by co-administration of Ang-(1-7). Interstitial and perivascular fibrosis in rats treated with Ang-(1-7) or with Dox/Ang-(1-7) was no different than in sham animals. The Dox-mediated increase in cardiac fibrosis and the anti-fibrotic effects of Ang-(1-7) co-administration may account for the changes in heart weight/body weight, since doxorubicin did not alter the size of cardiac myocytes. Cardiac function was assessed using a Vevo 2100 small animal ultrasound system, at baseline and following treatment. Doxorubicin reduced ejection fraction, fractional shortening and cardiac output compared to sham or Ang-(1-7) alone; co-administration of Ang-(1-7) with Dox tended to prevent the Dox-mediated reductions in cardiac contractility. The Dox-mediated increase in fibrosis may contribute to the reduced cardiac contractility and the Ang-(1-7)-mediated reduction in Dox-induced fibrosis may account for the improvement in contractility in rats co-administered Dox and Ang-(1-7). These results suggest that Ang-(1-7) may be effective in preventing Dox-induced cardiac fibrosis and may attenuate acute cardiac toxicity induced by Dox administration. Citation Format: Omeed A. Rahimi, Sharon M. Castellino, Cheryl E. Cammock, E Ann Tallant, Patricia E. Gallagher. Angiotensin-(1-7) prevents fibrosis in doxorubicin-treated rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4489. doi:10.1158/1538-7445.AM2015-4489

Abstract 72: Hydrogen Sulfide Prevents Myocardial Hypertrophy in a Klf5-dependent Manner

Rationale: H 2 S is a gasotransmitter that regulates multiple cardiovascular functions. Krüppel-like transcription factor (KLF) exerts diverse functions in the cardiovascular system. Objectives: The aim of present study was to investigate the effect of hydrogen sulfide (H 2 S) on myocardial hypertrophy. Methods and results: Myocardial samples of 22 patients with left ventricle hypertrophy were collected and underwent histological and molecular biological analysis. Spontaneously hypertensive rats (SHR) and neonatal rat cardiomyocytes were studied for functional and signaling response to GYY4137, a H 2 S-releasing compound. Expression of cystathionine -lyase (CSE), a main enzyme for H 2 S generation in human heart, decreased in human hypertrophic myocardium, while KLF5 expression increased. In SHR treated with GYY4137 for 4 weeks, myocardial hypertrophy was inhibited as evidenced by improvement in cardiac structural parameters, heart mass index, size of cardiac myocytes and expression of atrial natriuretic peptide (ANP). Levels of oxidative stress and phosphorylation of mitogen-activated protein kinases were also decreased after H 2 S treatment. H 2 S diminished expression of the KLF5 in myocardium of SHR and in neonatal rat cardiomyocytes rendered hypertrophy by angiotensin II (Ang II). H 2 S also inhibited ANP promoter activity and ANP expression in Ang II-induced neonatal rat cardiomyocyte hypertrophy, and these effects were suppressed by KLF5 knockdown. KLF5 promoter activity was increased by Ang II stimulation, and this was reversed by H 2 S. H 2 S also decreased activity of specificity protein-1 (SP-1) binding to the KLF5 promoter and attenuated KLF5 nuclear translocation by Ang II stimulation. Conclusion: H 2 S attenuated myocardial hypertrophy, which might be related to inhibiting oxidative stress and decreasing ANP transcription activity in a KLF5-dependent manner.

Optimum AT1 receptor-neprilysin inhibition has superior cardioprotective effects compared with AT1 receptor blockade alone in hypertensive rats

Neprilysin inhibitors prevent the breakdown of bradykinin and natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin II and endothelin-1. Here we studied the effects of a low and a high dose of the neprilysin inhibitor thiorphan on top of AT1 receptor blockade with irbesartan versus vehicle in TGR(mREN2)27 rats with high renin hypertension. Mean arterial blood pressure was unaffected by vehicle or thiorphan alone. Irbesartan lowered blood pressure, but after 7 days pressure started to increase again. Low- but not high-dose thiorphan prevented this rise. Only during exposure to low-dose thiorphan plus irbesartan did heart weight/body weight ratio, cardiac atrial natriuretic peptide expression, and myocyte size decrease significantly. Circulating endothelin-1 was not affected by low-dose thiorphan with or without irbesartan, but increased after treatment with high-dose thiorphan plus irbesartan. This endothelin-1 rise was accompanied by an increase in renal sodium-hydrogen exchanger 3 protein abundance, and an upregulation of constrictor vascular endothelin type B receptors. Consequently, the endothelin type B receptor antagonist BQ788 no longer enhanced endothelin-1-induced vasoconstriction (indicative of endothelin type B receptor-mediated vasodilation), but prevented it. Thus, optimal neprilysin inhibitor dosing reveals additional cardioprotective effects on top of AT1 receptor blockade in renin-dependent hypertension.

Epigenetic Therapy for the Treatment of Hypertension-Induced Cardiac Hypertrophy and Fibrosis.

The development of heart failure is associated with changes in the size, shape, and structure of the heart that has a negative impact on cardiac function. These pathological changes involve excessive extracellular matrix deposition within the myocardial interstitium and myocyte hypertrophy. Alterations in fibroblast phenotype and myocyte activity are associated with reprogramming of gene transcriptional profiles that likely requires epigenetic alterations in chromatin structure. The aim of our work was to investigate the potential of a currently licensed anticancer epigenetic modifier as a treatment option for cardiac diseases associated with hypertension-induced cardiac hypertrophy and fibrosis. The effects of DNA methylation inhibition with 5-azacytidine (5-aza) were examined in a human primary fibroblast cell line and in a spontaneously hypertensive rat (SHR) model. The results from this work allude to novel in vivo antifibrotic and antihypertrophic actions of 5-aza. Administration of the DNA methylation inhibitor significantly improved several echocardiographic parameters associated with hypertrophy and diastolic dysfunction. Myocardial collagen levels and myocyte size were reduced in 5-aza-treated SHRs. These findings are supported by beneficial in vitro effects in cardiac fibroblasts. Collagen I, collagen III, and α-smooth muscle actin were reduced in a human ventricular cardiac fibroblast cell line treated with 5-aza. These findings suggest a role for epigenetic modifications in contributing to the profibrotic and hypertrophic changes evident during disease progression. Therapeutic intervention with 5-aza demonstrated favorable effects highlighting the potential use of this epigenetic modifier as a treatment option for cardiac pathologies associated with hypertrophy and fibrosis.

The Ca(2+)-activated cation channel TRPM4 is a negative regulator of angiotensin II-induced cardiac hypertrophy.

Cardiac muscle adapts to hemodynamic stress by altering myocyte size and function, resulting in cardiac hypertrophy. Alteration in myocyte calcium homeostasis is known to be an initial signal in cardiac hypertrophy signaling. Transient receptor potential melastatin 4 protein (TRPM4) is a calcium-activated non-selective cation channel, which plays a role in regulating calcium influx and calcium-dependent cell functions in many cell types including cardiomyocytes. Selective deletion of TRPM4 from the heart muscle in mice resulted in an increased hypertrophic growth after chronic angiotensin (AngII) treatment, compared to WT mice. The enhanced hypertrophic response was also traceable by the increased expression of hypertrophy-related genes like Rcan1, ANP, and α-Actin. Intracellular calcium measurements on isolated ventricular myocytes showed significantly increased store-operated calcium entry upon AngII treatment in myocytes lacking the TRPM4 channel. Elevated intracellular calcium is a key factor in the development of pathological cardiac hypertrophy, leading to the activation of intracellular signaling pathways. In agreement with this, we observed significantly higher Rcan1 mRNA level, calcineurin enzyme activity and protein level in lysates from TRPM4-deficient mice heart compared to WT after AngII treatment. Collectively, these observations are consistent with a model in which TRPM4 is a regulator of calcium homeostasis in cardiomyocytes after AngII stimulation. TRPM4 contributes to the regulation of driving force for store-operated calcium entry and thereby the activation of the calcineurin–NFAT pathway and the development of pathological hypertrophy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-015-0501-x) contains supplementary material, which is available to authorized users.

Age-Related Features of Cardiomyocyte Ploidy in Patients with Hypertrophic Obstructive Cardiomyopathy.

Endomyocardial biopsy samples of the interventricular septum from patients with hypertrophic cardiomyopathy isolated during myectomy were examined. We revealed significant variability of cardiomyocyte ploidy (from 2.9c to 13.5c) that directly correlated with myocyte size; the maximum ploidy was found in young patients. Ultrastructural signs of increased contractile and synthetic activity of cells were observed in patients with high DNA content in cardiomyocytes.

Growth Dynamics and Mononucleation of the Ventricular Conduction System

During embryonic development, mammalian myocyte size does not change significantly. The switch from hyperplastic to hypertrophic growth occurs during the neonatal period. All previous studies have focused on working myocytes, which form 99% of ventricular mass; little is known about developmental changes in cell size, shape, and nucleation of cells of the ventricular conduction system (VCS). Hearts from Cx40:GFP transgenic mice were sampled at embryonic day 14, postnatal day 10, and adulthood (6 months). The ventricles were dissected, nuclei stained whole mount with Hoechst 33342, and specimens cleared in Scale/2 and flat mounted for confocal microscopy. Z‐series were acquired using a 40x oil immersion lens spanning at least 50 mm. Cell dimensions, nuclear count and volumes were measured separately for working and VCS myocytes. At the embryonic stage, green (Cx40‐expressing VCS cells) were larger than negative myocytes. This difference was reversed at postnatal day 10, where there was also differences in size between left bundle branch cells and Purkinje myocytes. The difference in cell volume was most pronounced at the adult stage. The transverse diameter was significantly smaller in adult VCS cells, giving them more elongated shape. Binucleated VCS cells were observed only rarely (<5%) during embryonic and neonatal stages; none (0/82) were seen in the adult. In conclusion, we show that the VCS in mouse is formed by a morphometrically distinct mononucleated cells with shape and size significantly different from working myocytesSupported by AAA travel grant and GACR P302/11/1308 and 13‐12412S.

SPARC Facilitates Inflammation in the Aging Heart and Suppresses Macrophage M2 Polarization

Cardiac aging is characterized by inflammation and fibrosis. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein known to play a role in extracellular matrix assembly and inflammation. SPARC levels increase with age in the left ventricle. We hypothesized that SPARC might facilitate age‐related cardiac inflammation. We studied 6 groups of mice (n=7‐10/group): young (3‐5 month old), middle‐aged (10‐12 m.o.) and old (18‐29 m.o.) C57BL/6J wild type (WT) and SPARC‐null (Null). Left ventricular wall thickness and myocyte size increased in an age‐related manner in WT but not Null (both p<0.05). Expression of pro‐inflammatory markers (Ccl5, Cx3cl1, Ccr2, Cxcr3) and Adamts1were increased in middle‐aged and old WT and old Null compared to respective young (all p<0.05). Versican is a substrate of Adamts1 that when cleaved promotes cell infiltration, and it decreased with age only in WT (p<0.05). Expression of α2 integrin and macrophage infiltration were increased with age in WT, but not Null. Isolated peritoneal macrophages from young and old WT showed similar levels of pro‐inflammatory M1 markers (Ccl5, Ccl3, Tnfα); however, peritoneal macrophages from old WT mice displayed reduced anti‐inflammatory M2 markers (Arg1, Mrc1). SPARC treatment of young macrophages decreased M2 markers to levels observed in old mice. In summary, SPARC mediates age‐related cardiac inflammation by suppressing M2 macrophage polarization.

Apoptosis, proliferation, and cell size in seasonal changes of body and organ weight in male bank voles Myodes glareolus

Adult male bank voles undergo the body and organ regression before winter, and in early spring, they resume the growth and reproductive processes. The aim of the present study was to determine whether the seasonal changes of body and organ weight in these animals depend on changes in the number of cells or their size. To study an autumnal regression, wild adult males captured in August were exposed to short photoperiod for 0, 4, and 8 weeks, while to study a spring resumption, overwintered males caught in March were exposed to long photoperiod for 0, 1, and 4 weeks. Apoptosis, proliferation, and cell size in the skeletal muscles, liver, and testes were examined. The study revealed that the seasonal changes of testes weight were associated with changes in the number of testicular cells. On the contrary, the changes in size of skeletal myocytes and hepatocytes appeared to be responsible for the seasonal changes of body (muscle) and liver weights in these rodents.

The orally active urotensin receptor antagonist, KR36676, attenuates cellular and cardiac hypertrophy.

Blockade of the actions of urotensin-II (U-II) mediated by the urotensin (UT) receptor should improve cardiac function and prevent cardiac remodelling in cardiovascular disease. Here, we have evaluated the pharmacological properties of the recently identified UT receptor antagonist, 2-(6,7-dichloro-3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-methyl-N-(2-(pyrrolidin-1-yl)-1-(4-(thiophen-3-yl)phenyl) ethyl)acetamide (KR36676). Pharmacological properties of KR36676 were studied in a range of in vitro assays (receptor binding, calcium mobilization, stress fibre formation, cellular hypertrophy) and in vivo animal models such as cardiac hypertrophy induced by transverse aortic constriction (TAC) or myocardial infarction (MI). KR36676 displayed high binding affinity for the UT receptor (Ki : 0.7 nM), similar to that of U-II (0.4 nM), and was a potent antagonist at that receptor (IC50 : 4.0 nM). U-II-induced stress fibre formation and cellular hypertrophy were significantly inhibited with low concentrations of KR36676 (≥0.01 μM). Oral administration of KR36676 (30 mg·kg(-1) ) in a TAC model in mice attenuated cardiac hypertrophy and myocardial fibrosis. Moreover, KR36676 restored cardiac function and myocyte size in rats with MI-induced cardiac hypertrophy. A highly potent UT receptor antagonist exerted anti-hypertrophic effects not only in infarcted rat hearts but also in pressure-overloaded mouse hearts. KR36676 could be a valuable pharmacological tool in elucidating the complicated physiological role of U-II and UT receptors in cardiac hypertrophy.

Adiponectin ameliorates hyperglycemia-induced cardiac hypertrophy and dysfunction by concomitantly activating Nrf2 and Brg1

Hyperglycemia-induced oxidative stress is implicated in the development of cardiomyopathy in diabetes that is associated with reduced adiponectin (APN) and heme oxygenase-1 (HO-1). Brahma-related gene 1 (Brg1) assists nuclear factor-erythroid-2-related factor-2 (Nrf2) to activate HO-1 to increase myocardial antioxidant capacity in response to oxidative stress. We hypothesized that reduced adiponectin (APN) impairs HO-1 induction which contributes to the development of diabetic cardiomyopathy, and that supplementation of APN may ameliorate diabetic cardiomyopathy by activating HO-1 through Nrf2 and Brg1 in diabetes. Control (C) and streptozotocin-induced diabetic (D) rats were untreated or treated with APN adenovirus (1×109 pfu) 3 weeks after diabetes induction and examined and terminated 1 week afterward. Rat left ventricular functions were assessed by a pressure–volume conductance system, before the rat hearts were removed to perform histological and biochemical assays. Four weeks after diabetes induction, D rats developed cardiac hypertrophy evidenced as increased ratio of heart weight to body weight, elevated myocardial collagen I content, and larger cardiomyocyte cross-sectional area (all P<0.05 vs C). Diabetes elevated cardiac oxidative stress (increased 15-F2t-isoprostane, 4-hydroxynonenal generation, 8-hydroxy-2′-deoxyguanosine, and superoxide anion generation), increased myocardial apoptosis, and impaired cardiac function (all P<0.05 vs C). In D rats, myocardial HO-1 mRNA and protein expression were reduced which was associated with reduced Brg1 and nuclear Nrf2 protein expression. All these changes were either attenuated or prevented by APN. In primarily cultured cardiomyocytes (CMs) isolated from D rats or in the embryonic rat cardiomyocytes cell line H9C2 cells incubated with high glucose (HG, 25 mM), supplementation of recombined globular APN (gAd, 2μg/mL) reversed HG-induced reductions of HO-1, Brg1, and nuclear Nrf2 protein expression and attenuated cellular oxidative stress, myocyte size, and apoptotic cells. Inhibition of HO-1 by ZnPP (10μM) or small interfering RNA (siRNA) canceled all the above gAd beneficial effects. Moreover, inhibition of Nrf2 (either by the Nrf2 inhibitor luteolin or siRNA) or Brg1 (by siRNA) canceled gAd-induced HO-1 induction and cellular protection in CMs and in H9C2 cells incubated with HG. In summary, our present study demonstrated that APN reduced cardiac oxidative stress, ameliorated cardiomyocyte hypertrophy, and prevented left ventricular dysfunction in diabetes by concomitantly activating Nrf2 and Brg1 to facilitate HO-1 induction.

Newborn hypoxia/anoxia inhibits cardiomyocyte proliferation and decreases cardiomyocyte endowment in the developing heart: role of endothelin-1.

In the developing heart, cardiomyocytes undergo terminal differentiation during a critical window around birth. Hypoxia is a major stress to preterm infants, yet its effect on the development and maturation of the heart remains unknown. We tested the hypothesis in a rat model that newborn anoxia accelerates cardiomyocyte terminal differentiation and results in reduced cardiomyocyte endowment in the developing heart via an endothelin-1-dependent mechanism. Newborn rats were exposed to anoxia twice daily from postnatal day 1 to 3, and hearts were isolated and studied at postnatal day 4 (P4), 7 (P7), and 14 (P14). Anoxia significantly increased HIF-1α protein expression and pre-proET-1 mRNA abundance in P4 neonatal hearts. Cardiomyocyte proliferation was significantly decreased by anoxia in P4 and P7, resulting in a significant reduction of cardiomyocyte number per heart weight in the P14 neonates. Furthermore, the expression of cyclin D2 was significantly decreased due to anoxia, while p27 expression was increased. Anoxia has no significant effect on cardiomyocyte binucleation or myocyte size. Consistently, prenatal hypoxia significantly decreased cardiomyocyte proliferation but had no effect on binucleation in the fetal heart. Newborn administration of PD156707, an ETA-receptor antagonist, significantly increased cardiomyocyte proliferation at P4 and cell size at P7, resulting in an increase in the heart to body weight ratio in P7 neonates. In addition, PD156707 abrogated the anoxia-mediated effects. The results suggest that hypoxia and anoxia via activation of endothelin-1 at the critical window of heart development inhibits cardiomyocyte proliferation and decreases myocyte endowment in the developing heart, which may negatively impact cardiac function later in life.

Revascularization of Chronic Hibernating Myocardium Stimulates Myocyte Proliferation and Partially Reverses Chronic Adaptations to Ischemia

BackgroundThe time course and extent of recovery after revascularization of viable dysfunctional myocardium are variable. Although fibrosis is a major determinant, myocyte structural and molecular remodeling may also play important roles. ObjectivesThis study sought to determine whether persistent myocyte loss and/or irreversibility of protein changes that develop in hibernating myocardium have an impact on functional recovery in the absence of infarction. MethodsSwine implanted with a chronic left anterior descending artery (LAD) stenosis to produce hibernating myocardium underwent percutaneous revascularization, with serial functional recovery evaluated for 1 month (n = 12). Myocardial tissue was evaluated to assess myocyte size, nuclear density, and proliferation indexes in comparison with those of normal animals and nonrevascularized controls. Proteomic analysis by 2-dimensional differential in-gel electrophoresis was used to determine the reversibility of molecular adaptations of hibernating myocytes. ResultsAt 3 months, physiological features of hibernating myocardium were confirmed, with depressed LAD wall thickening and no significant infarction. Revascularization normalized LAD flow reserve, with no immediate change in LAD wall thickening. Regional LAD wall thickening slowly improved but remained depressed 1 month post–percutaneous coronary intervention. Surprisingly, revascularization was associated with histological evidence of myocytes re-entering the growth phase of the cell cycle and increases in the number of c-Kit+ cells. Myocyte nuclear density returned to normal, whereas regional myocyte hypertrophy regressed. Proteomic analysis demonstrated heterogeneous effects of revascularization. Up-regulated stress and cytoskeletal proteins normalized, whereas reduced contractile and metabolic proteins persisted. ConclusionsDelayed recovery of hibernating myocardium in the absence of scar may reflect persistent reductions in the amounts of contractile and metabolic proteins. Although revascularization appeared to stimulate myocyte proliferation, the persistence of small immature myocytes may have contributed to delayed functional recovery.

Gene expression changes controlling distinct adaptations in the heart and skeletal muscle of a hibernating mammal.

Throughout the hibernation season, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) experiences extreme fluctuations in heart rate, metabolism, oxygen consumption, and body temperature, along with prolonged fasting and immobility. These conditions necessitate different functional requirements for the heart, which maintains contractile function throughout hibernation, and the skeletal muscle, which remains largely inactive. The adaptations used to maintain these contractile organs under such variable conditions serves as a natural model to study a variety of medically relevant conditions including heart failure and disuse atrophy. To better understand how two different muscle tissues maintain function throughout the extreme fluctuations of hibernation we performed Illumina HiSeq 2000 sequencing of cDNAs to compare the transcriptome of heart and skeletal muscle across the circannual cycle. This analysis resulted in the identification of 1,076 and 1,466 differentially expressed genes in heart and skeletal muscle, respectively. In both heart and skeletal muscle we identified a distinct cold-tolerant mechanism utilizing peroxisomal metabolism to make use of elevated levels of unsaturated depot fats. The skeletal muscle transcriptome also shows an early increase in oxidative capacity necessary for the altered fuel utilization and increased oxygen demand of shivering. Expression of the fetal gene expression profile is used to maintain cardiac tissue, either through increasing myocyte size or proliferation of resident cardiomyocytes, while skeletal muscle function and mass are protected through transcriptional regulation of pathways involved in protein turnover. This study provides insight into how two functionally distinct muscles maintain function under the extreme conditions of mammalian hibernation.

The alteration of protein prenylation induces cardiomyocyte hypertrophy through Rheb-mTORC1 signalling and leads to chronic heart failure.

G protein-regulated cell function is crucial for cardiomyocytes, and any deregulation of its gene expression or protein modification can lead to pathological cardiac hypertrophy. Herein, we report that protein prenylation, a lipidic modification of G proteins that facilitates their association with the cell membrane, might control the process of cardiomyocyte hypertrophy. We found that geranylgeranyl diphosphate synthase (GGPPS), a key enzyme involved in protein prenylation, played a critical role in postnatal heart growth by regulating cardiomyocyte size. Cardiac-specific knockout of GGPPS in mice led to spontaneous cardiac hypertrophy, beginning from week 4, accompanied by the persistent enlargement of cardiomyocytes. This hypertrophic effect occurred by altered prenylation of G proteins. Evaluation of the prenylation, membrane association and hydrophobicity showed that Rheb was hyperactivated and increased mTORC1 signalling pathway after GGPPS deletion. Protein farnesylation or mTORC1 inhibition blocked GGPPS knockdown-induced mTORC1 activation and suppressed the larger neonatal rat ventricle myocyte size and cardiomyocyte hypertrophy in vivo, demonstrating a central role of the FPP-Rheb-mTORC1 axis for GGPPS deficiency-induced cardiomyocyte hypertrophy. The sustained cardiomyocyte hypertrophy progressively provoked cardiac decompensation and dysfunction, ultimately causing heart failure and adult death. Importantly, GGPPS was down-regulated in the hypertrophic hearts of mice subjected to transverse aortic constriction (TAC) and in failing human hearts. Moreover, HPLC-MS/MS detection revealed that the myocardial farnesyl diphosphate (FPP):geranylgeranyl diphosphate (GGPP) ratio was enhanced after pressure overload. Our observations conclude that the alteration of protein prenylation promotes cardiomyocyte hypertrophic growth, which acts as a potential cause for pathogenesis of heart failure and may provide a new molecular target for hypertrophic heart disease clinical therapy.

Omentin functions to attenuate cardiac hypertrophic response

Cardiac hypertrophy occurs in many obesity-related conditions. Omentin is an adipose-derived plasma protein that is downregulated under obese conditions. Here, we investigated whether omentin modulates cardiac hypertrophic responses in vivo and in vitro. Systemic administration of an adenoviral vector expressing human omentin (Ad-OMT) to wild-type (WT) mice led to the attenuation of cardiac hypertrophy, fibrosis and ERK phosphorylation induced by transverse aortic constriction (TAC) or angiotensin II infusion. In cultured cardiomyocytes, stimulation with phenylephrine (PE) led to an increase in myocyte size, which was prevented by pretreatment with human omentin protein. Pretreatment of cardiomyocytes with omentin protein also reduced ERK phosphorylation in response to PE stimulation. Ad-OMT enhanced phosphorylation of AMP-activated protein kinase (AMPK) in the heart of WT mice after TAC operation. Blockade of AMPK activation by transduction with dominant-negative mutant forms of AMPK reversed the inhibitory effect of omentin on myocyte hypertrophy and ERK phosphorylation following PE stimulation. Moreover, fat-specific transgenic mice expressing human omentin showed reduced cardiac hypertrophy and ERK phosphorylation following TAC surgery compared to littermate controls. These data suggest that omentin functions to attenuate the pathological process of myocardial hypertrophy via the activation of AMPK in the heart, suggesting that omentin may represent a target molecule for the treatment of cardiac hypertrophy.

Abstract 15308: SPARC Deletion Suppresses Age-related Cardiac Inflammation

Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein that facilitates the formation and assembly of mature cross-linked collagen fibrils. We previously reported that aged mice exhibit increased SPARC levels in the heart, and SPARC deletion inhibits age-related collagen deposition. The present study investigated whether SPARC deletion could suppress age-related cardiac inflammation. Six groups of mice were studied: young (3-5 month old), middle-aged (10-12 m.o.) and old (18-29 m.o.) C57BL/6J wild type (WT) and SPARC-null (Null) mice (n=5-10 per group). Echocardiography and histology revealed that LV wall thickness and myocyte size were 15% and 43% increased with age in WT, respectively (both p&lt;0.05 old versus young). These age-dependent structural changes were blunted in the Null. The pro-inflammatory chemokines Ccl5 and Ccr2 were increased in middle-aged and old WT, and Cxcl1 was increased in old WT, compared to young WT (all p&lt;0.05). Interestingly, the age-dependent increases in Ccl5, Ccr2, and Cxcl1 were delayed in Null. Migration inhibitory factor (Mif) and Cx3cl1 showed an age-dependent increase in both WT and Null. Cxcl5 expression levels increased age-dependently only in Null, suggesting a compensatory increase for the decline of other chemokines. In summary, these results suggest that SPARC deletion slowed age-related cardiac inflammation.

Abstract 18651: Osteopontin RNA Aptamer Reverses Heart Failure and Increases Plasma HDL Levels

Objective: Osteopontin (OPN) expression increases in the heart during hypertrophy and heart failure and in the aorta during atherosclerosis. OPN KO mice were reported to have a reduced hypertrophic response after trans-aortic constriction (TAC), and a reduced plaque burden on an APOE-/- background. We hypothesized that an RNA aptamer targeting OPN protein would effectively modulate the responses to TAC and atherogenesis. Method and Results: C57BL6 male mice were subjected to sham or TAC surgeries. TAC mice were injected starting day1 with OPN RNA aptamer or mutant OPN aptamer. At 4wks, OPN aptamer treatment increased %EF (25%) relative to mut control (n=13, p&lt;.01). This significant cardiac improvement was sustained at 8 wks (n=7) and 12wks (n=4-7). WGA and PR staining showed respectively that the OPN aptamer significantly reduced myocyte size (.8 fold at 4wks and 0.7 fold at 12 wks) and cardiac fibrosis (0.4 fold at 4wks and 0.3 fold at 12 wks). In 4 and 12wk LVs and RVs, OPN aptamer reduced Nppb, Nppa, Opn, Fn1, Col3a1, and Lum transcripts (n=3-6 - p&lt;.05). At 12 wk, FN1 protein was reduced in the LVs and RVs by the OPN Apt (n=3 p&lt;.05) and serum HDL was increased (20% p=.001; n=5-7). In 6 mo APOE-/- mice, systemic injections of OPN aptamer also increased plasma HDL (13%; p=.02; n=8-10).To assess effect of OPN aptamer on reversal of cardiac remodeling, at 8 wks post TAC (n=3-4), cardiac function was assessed followed by a total of 7 OPN aptamer injections (one every other day), and then assessed again at 10 wks. The OPN aptamer improved %EF in TAC mice (.6 fold p=.02 TAC vs Sham before treatment to .85 fold p=NS after treatment). In wild type but not OPN KO neonatal mouse cardiomyocytes-fibroblast co-culture, 16h treatment with FAM-labeled OPN aptamer, followed by Phalloidin and TNT staining, showed successful intracellular uptake of the OPN aptamer by cardiac myocytes and more so by fibroblasts. Conclusion: OPN RNA aptamer protected against and reversed cardiac dysfunction in a mouse TAC model of cardiac hypertrophy, and increased serum HDL in WT and APOE-/- mice. The cardioprotective effect of the OPN aptamer, together with the efficient uptake by cardiac myocytes and fibroblasts make it a promising therapeutic molecule and a potential platform for targeted RNA delivery into the heart.

Abstract 17939: Lack of the Alpha-11 Integrin in the Heart is Associated With Progressive Diastolic Dysfunction, Myofibrillar Disarray and Impaired Cardiomyocyte Growth

BACKGROUND: Integrins, transmembrane receptors, play crucial roles in diverse cellular and developmental processes due to critical interactions with the extracellular matrix (ECM). During fetal development and towards adulthood, heart growth and function is suggested to depend on forming and remodeling the ECM and its connection to the myocyte. Currently however, the role of integrins in cardiovascular development (CVD) is poorly defined. Thus, we hypothesized that the α11 integrin (α11), which is expressed by fibroblasts and binds preferentially to type I collagen fibers, plays a vital role in CVD. METHODS: α11 KO and wildtype littermate mice (both n = 8) were examined at 4 weeks and 8 weeks of age. Animals underwent function assessments, including echocardiography and invasive pressure volume (PV) loop analysis, and structural examination via histological and electron microscopy (EM) analysis. RESULTS: At 4 weeks, heart weight (HW) and HW indexed to tibial length were decreased in α11 KO mice (P &lt; 0.05), which were normalized at 8 weeks. Echocardiography revealed reduced end-diastolic area (EDA) at 4 weeks (P &lt; 0.05). Despite normalization of EDA at 8 weeks, PV loop revealed impaired diastolic function as evidence by increased EDP, prolonged Tau and steeper EDPVR (all P &lt; 0.05). No differences in HR or systolic parameters were evident. α11 KO mice also demonstrated structural changes. WGA staining revealed evidence of myofibrillar disarray. Connexin 43 and desmin staining showed increased Z-disk and intermediate filament clustering, respectively. LV myocyte size was also reduced (P &lt; 0.05). Similarly, EM analysis showed reduced cardiomyocyte thickness and distance between end plates (both P &lt; 0.05). CONCLUSION: Loss of α11 resulted in progressively worsening diastolic function that was associated with myofibrillar disarray and impaired cardiomyocyte growth. These findings suggest that α11 is required for the development of normal heart structure and function.