Actomyosin Function in Left and Right Ventricles of Failing Human Hearts is Identical

Review Article

Austin Cardiol. 2017; 2(2): 1011.

Actomyosin Function in Left and Right Ventricles of Failing Human Hearts is Identical

Borejdo J¹*, Nagwekar J¹, Duggal D¹, Raut S², Rich R³, Fudala R¹, Das H4, Gryczynski Z2 and Gryczynski I¹

¹Department of Cell Biology and Center for Commercialization of Fluorescence Technologies, University of North Texas, USA

²Department of Physics and Astronomy, Texas Christian University, USA

³Department of Mathematics and Physics, Texas Wesleyan University, USA

4Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas, USA

*Corresponding author: Julian Borejdo, Department of Cell Biology, University of North Texas Health Science Center, USA

Received: September 25, 2017; Accepted: October 20, 2017; Published: October 27, 2017


Human systemic blood system offers larger resistance to the blood flow than pulmonary system: the Left Ventricle (LV) pumps blood more forcefully than the Right Ventricle (RV). The difference in pumping action may arise from the more efficient interaction between actin and myosin in the LV, from the more efficient operation of other sarcomericproteins or from the morphological differences between ventricles. These questions cannot be answered by using whole ventricles or isolated myocytes because the number of molecules involved in tension generation is of the order of 1011. Averaging data from such large number of molecules makes unequivocal characterization of any differences impossible. Measurements must be taken from a few molecules. Moreover, data must be obtained in-situ to account for the molecular crowding effects that are likely to occur in crowded environment such as muscle. We measured kinetics of actomyosin cycle in contracting myocytes from failing ventricles by analyzing fluctuations in orientation of a few actin and myosin molecules in situ. Fluctuations in orientation were caused by ATP-induced repetitive cycles of binding-dissociation of myosin from actin. In both left and right failing ventricles the rate constants characterizing contraction were identical. We also measured distribution of spatial orientations of actin and myosin. The spatial distributions were identical in myocytes from both ventricles.These results show that there is no difference in the way actomyosin interacts with thin filaments in failing left and right ventricles, suggesting that the difference in pumping efficiencies are due either to muscle proteins other than actin and myosin, or that they are due to morphological differences between left and right ventricles.

Keywords: Cross-bridge orientation; Heart ventricles; Fluorescence polarization


ACF: Auto Correlation Function; AP: Alexa633 Phalloidin; EDC: Ethyl-3-[3-(dimethylamino)-propyl]-Carbodiimide; FCS: Fluorescence Correlation Spectroscopy; LV: Left Ventricle; HF: Ventricles from the Failing Heart; NF: Ventricles from Non Failing Heart; OV: Observational Volume; PF: Polarization of Fluorescence; RV: Right Ventricle; SD: Standard Deviation; SSA: Steady State Anisotropy; UP: Unlabeled Phalloidin; XB: Myosin Cross-Bridge


The Left Ventricle (LV) has to overcome a large resistance offered by a systemic system, while the Right Ventricle (RV) has to overcome a lesser resistance offered by a pulmonary system. However, it is not clear whether the ability to develop larger force by the LV is due to: 1. More efficient force generation by actin and myosin, 2. More efficient operation of other muscle proteins or 3. Dissimilarities of basic fiber structures of the two ventricles. The left ventricle is composed largely of oblique and circumferential fibers [1], which are known to be more mechanically efficient than the transverse fibers in the free wall of the right ventricle [2,3].

Orientation of the lever arm of myosin head is a defining parameter of force producing interaction between actin and myosin. In order to expose differences in this interaction between left and right ventricles, it is necessary to measure the rate constants governing the interaction, and the spatial distribution of myosin lever arm orientations associated with contraction of each ventricle. Whole ventricles or isolated myocytes cannot be used in such experiments because they contain millions of actomyosin molecules and data originating from so many molecules would get averaged out. All the rate constants of the mechanochemical cycle of actomyosin become unrecoverable, and the final distribution of orientations a large assembly will be a perfect Gaussian, irrespective of whether the data are taken from the left or right ventricle (Central Limit Theorem, [4]). In assessing the actin-myosin interaction, the contribution of individual molecules has to be measured [5,6]. Because of these technical difficulties the question whether individual actin and myosin molecules of LV and RV interact differently has never been asked.

We developed the ability to study few molecules out of millions present in a myocyte. This was possible by focusing on a minute section of a sarcomere. Force-producing interactions between actin and myosin take place the Overlap-band (O-band). We measured kinetics and spatial distribution of a few molecules of actin and myosin in the O-bandof sarcomere inworking failing myocytes. It is important that the measurements be carried in-situ because protein concentration in muscle is high, [7]). Consequently the molecular crowding effects mayplay a role in the operation of muscle [8,9]. We measured fluctuations of orientation of a few actomyosin molecules in situ during contraction. Fluctuations were reported by anisotropy of fluorescence, which is a convenient method to measure conformation changes [10-15]. From fluctuations we calculated the rate constants of myosin interacting with actin.We also compared the spatial distribution of actin and myosin molecules during contraction of both ventricles.

The results show that the kinetics and the steady-state distribution of actin and myosin were the same in contracting myocytes from left and right ventricles from failing human heart It follows that the difference in ventricular function are caused either by non-tension generating muscle proteins or by morphological differences between ventricles.

Materials and Methods

Chemicals and solutions

All chemicals were from Sigma-Aldrich (St Louis, MO) except the fluorescent dye SeTau-647-mono-maleimide which was from SETA BioMedicals (Urbana, IL) and Alexa633 phalloidin (AP) and Unlabeled Phalloidin (UP) were from Molecular Probes (Eugene, OR). The glycerinating solution contained: 50% glycerol, 150 mMKCl, 10mM Tris-HCl pH 7.5, 5 mM MgCl2, 5 mM EGTA, 5 mM ATP, 1 mM DTT, 2 mM PMSF and 0.1% β-mercaptoethanol. Carigor solution contained 50 mMKCl, 10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 0.1 mM CaCl2. Contracting solution contained in addition 5 mM ATP, 20 mMcreatine phosphate and 10 units/ml of 1 mg/ml creatine kinase. EDTA-rigor solution contained 50 mMKCl, 10 mM Tris-HCl pH 7.5, 5 mM EDTA. Glycerinating solution contained 50 mMKCl, 10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 2 mM EGTA, 5 mM ATP, 20 mMcreatine phosphate and 10 units/ml of 1mg/ml creatine kinase.

Preparation of ventricles

Samples of human myocardium were collected at the University of Kentucky using procedures that were approved by the local Institutional Review Board. Failing myocardial samples were procured from patients who received heart transplants. All samples were passed to a researcher as soon as they were removed from the patient and snap-frozen in liquid nitrogen within a few minutes. Samples were shipped to UNTHSC on dry ice. Immediately upon arrival in Fort Worth they placed for 24 hrs in glycerinating solution at 0ºC. After 24 hrs, the glycerinating solution was replaced with a fresh solution and placed at -20ºC. Myocytes (MF) were made from glycerinated hearts after a minimum of 2 weeks at -20ºC.

Preparation of myocytes

2 weeks in a solution containing glycerine and EGTAcaused creation of large holes in the myocyte membrane allowing direct access of MgATP to the O-bands. Myocytes are only ~0.5μm thick thus limiting the Observational Volume to AttoLiters (10-12L, see Figure 2A). Preparation of myocytes involved thorough washing of ventricles with ice-cold EDTA-rigor solution and incubating them for 1hr in this solution in order to wash out ATP present in the glycerinating solution without causing contraction. They were then washed thoroughly with Ca-rigor solutionand homogenized in the Cole-Palmer LabGen 125 homogenizer for 10s followed by further 10s homogenization after a cool down period of 30s. Myocytes were made from ventricles which had spent at least 2 weeks in glycerinating solution at -20ºC, and were used within 2 days of preparation.