Alpha-myosin Heavy Chain Expression Research Articles

Abstract 614: Using Intact Trabeculae to Determine the Effect of Myosin-Modifying Drugs on Work, Power, and Mechanical Control of Relaxation

Background: Heart failure and especially heart failure with preserved ejection fraction are significant burdens on health. Intact cardiac trabeculae may reveal functional changes that cannot be seen in other ex vivo (skinned, single molecule) preparations. For example, using intact trabeculae, we have shown that relaxation is mechanically controlled by the lengthening strain rate at end systole, not afterload. Objective: We sought to evaluate the effect of myosin activator Omecamtiv Mercarbil (OM) and inhibitor Mavacamten (Mav) on physiologic function in intact trabeculae. Methods/Results: Afterload-clamp protocols were applied to intact cardiac trabeculae from Sprague Dawley rats to simulate physiologic work-loops and evaluate mechanical control of relaxation. Both OM and Mav reduced stroke work (force x length) by >50% and power (force x velocity) by ~50% at doses reducing developed force by 50%. These were mediated by dose-dependent reductions in both force and shortening length. We have recently reported preliminary results that OM improves contraction-relaxation coupling and makes the relaxation rate more sensitive to strain rate in a dose-dependent manner. Mav does not lead to significant changes in contraction-relaxation coupling at any dose; Mav alters the sensitivity of relaxation rate to strain rate only at doses that reduce developed force by 50%. Summary/Perspective: Intact rat cardiac trabeculae reveal function mimicking physiology. OM and Mav show remarkable similarities in force, work, and power, which may be due to the high expression of alpha-myosin heavy chain. OM treatment not only modified contractility but increased sensitivity of the relaxation rate to strain rate in a dose dependent manner, which may explain why diastolic dysfunction is not more prevalent in clinical studies. Mav appears to only modify the attachment of crossbridges as expected.

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.

Variable optimization for the formation of three-dimensional self-organized heart muscle

Cardiac tissue-engineering research is focused on the development of functional three-dimensional (3D) heart muscle in vitro. These models allow the detailed study of critical events in organogenesis, such as the establishment of cell-cell communication and construction and modification of the extracellular matrix. We have previously described a model for 3D heart muscle, termed cardioids, formed by the spontaneous delamination of a cohesive monolayer of primary cells in the absence of any synthetic scaffolding material. In an earlier publication, we have shown that, upon electrical stimulation, cardioids generate a twitch force in the range of 200-300 microN, generate a specific force (twitch force normalized to total cross-sectional area) of 2-4 kN/m(2), and can be electrically paced at frequencies of up to 10 Hz without any notable fatigue. We have two objectives for the current study: model development and model optimization. Our model development efforts are focused on providing additional characterization of the cardioid model. In this study, we show for the first time that cardioids show a pattern of gene expression comparable to that of cells cultured in two dimensions on tissue culture plastic and normal mammalian heart muscle. Compared with primary cardiac cells cultured on tissue culture plastic, the expression of alpha-myosin heavy chain (MHC), beta-MHC, SERCA2, and phospholamban was significantly higher in cardioids. Our second objective, model optimization, is focused on evaluating the effect of several cell culture variables on cardioid formation and function. Specifically, we looked at the effect of plating density (1.0-4.0 x 10(6) cells per cardioid), concentration of two adhesion proteins (laminin at 0.2-2.0 microg/cm(2) and fibronectin at 1-10 microg/cm(2)), myocyte purity (using preplating times of 15 and 60 min), and ascorbic acid stimulation (1-100 microl/ml). For our optimization studies, we utilized twitch force in response to electrical stimulation as our endpoint metric. Based on these studies, we found that cardioids formed with a plating density in the range 3-4 x 10(6) cells per cardioid generated the maximum twitch force, whereas increasing the surface adhesion protein (using either laminin or fibronectin) and increasing the myocyte purity both resulted in a decrease in twitch force. In addition, increasing the ascorbic acid concentration resulted in an increase in the baseline force of cardioids, which was recorded in the absence of electrical stimulation. Based on the model development studies, we have shown that cardioids do indeed exhibit a gene expression pattern similar to normal mammalian heart muscle. This provides further validity for the cardioid model. Based on the model optimization studies, we have identified specific cell culture regimes which support cardioid formation and function. These results are specific to the cardioid model; however, they may be translated and applied to other tissue-engineering models. Collectively, the work described in this study provides insight into the formation of functional 3D heart muscle and the effect of several cell culture variables on tissue formation and function.

Reduced Expression of Alpha MHC in Failing, Pre-LVAD Human Myocardium Contributes to Depressed Rates of ATP Utilization

The ventricles of human myocardium normally express low levels of α myosin heavy chain (MHC) on a predominately β MHC background. However, in heart failure the distribution changes to ∼100% β MHC with virtually undetectable levels of α MHC. While it has been known for some time that α MHC exhibits greater rates of ATP utilization and maximal shortening velocity (Vmax), we have recently shown that the low level of α MHC normally present in the ventricles of larger mammals increases the rate of rise of force compared to myocardium expressing 100% β MHC. Here, we tested the hypothesis that the loss of α MHC in human heart failure impairs contraction kinetics and contributes to mechanical dysfunction by measuring the rate of ATP utilization and isometric force in normal donor hearts and in failing myocardium excised from patients prior to the implantation of a left ventricular assist device (LVAD). Permeabilized multicellular preparations from normal myocardium yielded maximal rates of ATP turnover approximately 3-fold greater than in pre-LVAD failing myocardium, while maximal isometric force between the two groups was similar. This equates to a nearly 3-fold greater tension cost in normal human myocardium, and it is possible that the lower tension cost observed in failing myocardium would enhance the efficiency of contraction under conditions of impaired energetics. Furthermore, SDS-PAGE indicated a reduction in α MHC content in pre-LVAD, failing myocardium compared to normal myocardium. These results suggest that a loss of α MHC in human heart failure would be at least partly responsible for the decrease in contractile function and would contribute to lower rates of pressure development (dP/dt) in vivo, ultimately impairing both systole and diastole. This work supported by NIH RO1-HL61635 (RLM).

MicroRNA-1 and MicroRNA-133 in Spontaneous Myocardial Differentiation of Mouse Embryonic Stem Cells

MicroRNAs (miRNAs) regulate various biological processes through inhibiting the translation of RNA transcripts. Although miRNA-1 (miR-1) and miRNA-133 (miR-133) are abundantly expressed in the adult heart and involved in cardiac hypertrophy, the roles of these miRNAs in spontaneous myocardial differentiation are unknown. The levels of miR-1 and miR-133 in mouse embryonic stem (ES) cells were increased during spontaneous differentiation by 2-dimensional culture, but reduced during forced myocardial differentiation by a histone deacetylase inhibitor, trichostatin A. The overexpression of miR-1 or miR-133 by lentiviral infection reduced the expression of a cardiac-specific gene, Nkx2.5, during differentiation of ES cells. In addition, miR-1 also inhibited alpha-myosin heavy chain expression. The results of luciferase assays revealed that miR-1 recognizes and targets the 3' untranslated region of cyclin-dependent kinase-9 (Cdk9) in ES cells. Overexpression of miR-1 decreased the protein amounts of Cdk9 without affecting the mRNA levels, indicating that miR-1 post-transcriptionally inhibits Cdk9 translation. miR-1 and miR-133 may play significant roles in the myocardial differentiation of mouse ES cells, and Cdk9 may be involved in this process as a target of miR-1.

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.

Overt expression of AP-1 reduces alpha myosin heavy chain expression and contributes to heart failure from chronic volume overload

Reduced expression of α-MHC plays a significant role in cardiac contractile dysfunction from hemodynamic overload. Previously, Pur proteins and YY1 have been shown to play a role in α-MHC repression during heart failure induced by pressure overload and by spontaneous hypertension, respectively. This was not observed in volume-overload-induced heart failure, suggesting additional regulatory mechanisms for α-MHC repression. The present study was performed to identify volume overload responsive transcription factors involved in α-MHC gene regulation. DNA binding activity of several transcription factors was evaluated in a functionally characterized rat model of heart failure induced by aorto-caval shunt. After 10 weeks of shunt, severe LV dilatation and reduced LV function were accompanied by increased expression of ANF and β-MHC, and decreased expression of α-MHC. This was associated with dramatic (10-fold) activation of AP-1 together with increased expression of c-fos and c-jun. AP-1 activation was not observed following 4 weeks of shunt when cardiac function was preserved. In cultured cardiomyocytes, induction of AP-1 by PMA attenuated α-MHC mRNA by 60%. Transient transfection assays mapped PMA responsive sequence to − 582 to − 588 bp of α-MHC promoter. Deletion or mutation of these nucleotides had minimal effect on basal promoter activity but played a dominant role in PMA-mediated repression of α-MHC promoter activity. Over-expression of c-fos and c-jun in cardiomyocytes inhibited α-MHC promoter activity in a concentration dependent manner. Data suggest a repressive role of AP-1 in α-MHC expression and its possible involvement in the transition from compensatory hypertrophy to heart failure in chronic volume overload.

The estrogen receptor-α agonist 16α-LE2 inhibits cardiac hypertrophy and improves hemodynamic function in estrogen-deficient spontaneously hypertensive rats

Cardiac mass increases with age and with declining estradiol serum levels in postmenopausal women. Although the non-selective estrogen receptor-alpha and -beta agonist 17beta-estradiol attenuates cardiac hypertrophy in animal models and in observational studies, it remains unknown whether activation of a specific estrogen receptor subtype (ERalpha or ERbeta) might give similar or divergent results. Therefore, we analyzed myocardial hypertrophy as well as cardiac function and gene expression in ovariectomized, spontaneously hypertensive rats (SHR) treated with the subtype-selective ERalpha agonist 16alpha-LE2 or 17beta-estradiol. Long-term administration of 16alpha-LE2 or 17beta-estradiol did not affect elevated blood pressure, but both agonists efficiently attenuated cardiac hypertrophy and increased cardiac output, left ventricular stroke volume, papillary muscle strip contractility, and cardiac alpha-myosin heavy chain expression. The observed effects of E2 and 16alpha-LE2 were abrogated by the ER antagonist ZM-182780. Improved left ventricular function upon 16alpha-LE2 treatment was also observed in cardiac MRI studies. In contrast to estradiol and 16alpha-LE2, tamoxifen inhibited cardiac hypertrophy but failed to increase alpha-myosin heavy chain expression and cardiac output. These results support the hypothesis that activation of ERalpha favorably affects cardiac hypertrophy, myocardial contractility, and gene expression in ovariectomized SHR. Further studies are required to determine whether activation ERbeta mediates redundant or divergent effects.

P4-02 Cardiac mRNA gene expression of alpha-myosin heavy chain is decreased and beta-myosin heavy chain is increased in both ischemic and idiopathic dilated cardiomyopathy

P4-02 Cardiac mRNA gene expression of alpha-myosin heavy chain is decreased and beta-myosin heavy chain is increased in both ischemic and idiopathic dilated cardiomyopathy

The effect of angiotensin II on myosin heavy chain expression in cultured myocardial cells.

Angiotensin II (AII), the principal mediator of the renin-angiotensin system, is an important regulator of vascular and cardiac homeostasis. AII has also been shown to be a regulator of cardiac hypertrophy and of the corresponding changes in amount and composition of certain tissue proteins. We examined the trophic effects of AII on cultured myocytes derived from neonatal rat ventricles and followed, by Northern blot analysis and polyacrylamide gel electrophoresis, the expression of alpha- and beta-myosin heavy chain iso-mRNAs and isoproteins. Our findings show that a single administration of AII is sufficient to induce a trophic response in cultured beating myocytes and to enhance the expression of beta-myosin heavy chain iso-mRNA and isoprotein, having no effect on alpha-myosin heavy chain. Induction of alpha-myosin heavy chain expression by thyroid hormone before AII was administered showed that AII could not potentiate a shift from alpha- to beta-myosin heavy chain predominance. We suggest that the potency of AII to regulate the expression of myosin heavy chain isogenes is restricted to the beta isoform and is overridden by thyroid hormone.

Molecular and cellular biology of the senescent hypertrophied and failing heart

During aging, experimental studies have revealed various cellular changes, principal among which is myocyte hypertrophy, which compensates for the loss of myocytes and is associated with fibrosis. The expression of alpha-myosin heavy chain is replaced by that of the isogene beta-myosin, which leads to decreased myosin adenosine triphosphatase (ATPase) activity. In consequence, contraction is slower and more energetically economical. The Ca(2+)-ATPase of the sarcoplasmic reticulum and Na+/Ca2+ exchange activity are decreased, which probably explains the reduced velocity of relaxation. Membrane receptors are also modified, since the density of both the total beta-adrenergic and muscarinic receptors is decreased. The senescent heart is able to hypertrophy in response to overload and to adapt to the new requirements. Similar alterations are observed both in the senescent heart and in the overloaded heart, in clinical as well as in experimental studies; however, differences do exist, especially in terms of fibrosis and arrhythmias.

The influence of cold stress on the myosin heavy chain expression of cardiac and smooth muscle in normotensive and spontaneously hypertensive female rats.

Cold exposure (6 weeks at 4 degrees C) of normotensive (Wistar-Kyoto) and stroke-prone spontaneously hypertensive female rats led to cardiac hypertrophy (in stroke-prone spontaneously hypertensive rats), increased the level of plasma thyroxine, and increased the alpha-myosin heavy chain expression in the left ventricle. In contrast, myosin heavy chain expression of both main mesenteric artery and uterus was not affected by cold stress and chronic hypertension, suggesting different regulation of myosin heavy chain expression in smooth and cardiac muscle in vivo.

Isomyosin expression patterns during rat heart morphogenesis: an immunohistochemical study.

An immunohistochemical study of cardiac alpha and beta myosin heavy chain (MHC) expression during rat heart morphogenesis was performed. In tubular hearts (embryonic days, ED10-11) coexpression of both cardiac alpha and beta MHC was found throughout the heart, except for the left free wall of the atrium, where only cardiac alpha MHC is detected. A transition of coexpression to single expression of either cardiac alpha or beta MHC begins at the same time in both atria and ventricles but requires a longer time for completion in the ventricules; in the atria transition takes place during the period ED 12-13 and in the ventricles during ED12-15. Furthermore, expression of cardiac alpha and beta MHC was detected in the sinus venosus, and cardiac alpha MHC expression was detected in the pulmonary veins. A comparison of the results obtained in chicken embryos revealed that in tubular hearts the expression pattern is similar, whereas in later developmental stages two major differences were observed: 1) transition of coexpression to single expression in rat ventricles appears to take a longer developmental period; 2) the persistence of areas of coexpression in the sinoatrial junction, dorsal mesocardium, atrioventricular junction, and outflow tract, as found in the chicken embryo in later developmental stages, is not found in the rat heart.