Articles in PresS. Am J Physiol Heart Circ Physiol (September 26, 2014). doi:10.1152/ajpheart.00423.2014

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Effects of a Myofilament Calcium Sensitizer on Left Ventricular Systolic and Diastolic Function

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in Rats with Volume Overload Heart Failure

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Kristin Wilson1,2, Anuradha Guggilam1, T. Aaron West1, Xiaojin Zhang1, Aaron J. Trask1,3,

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Mary J. Cismowski1,3, Pieter de Tombe4, Sakthivel Sadayappan4, Pamela A. Lucchesi1,3*

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Children’s Hospital, Columbus, OH; 2Department of Veterinary Biosciences, College of

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Veterinary Medicine, and 3Department of Pediatrics, College of Medicine, The Ohio State

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University, Columbus OH; and 4Department of Cellular and Molecular Physiology, Stritch

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School of Medicine, Loyola University Chicago, Maywood, IL

Center for Cardiovascular and Pulmonary Research and The Heart Center, Nationwide

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*Corresponding Author:

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Pamela A. Lucchesi, PhD, FAHA Director, Center for Cardiovascular and Pulmonary Research The Research Institute at Nationwide Children's Hospital 575 Children's Crossroads, WB-4157 Columbus, OH 43215 Phone (614) 355-5753 Fax (614) 355-5726 [email protected]

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Figures 10

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Tables 1

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Words 7437 (with references)

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Copyright © 2014 by the American Physiological Society.

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Abstract

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Background. Aortocaval fistula (ACF)-induced volume overload (VO) heart failure (HF) results

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in progressive left ventricular (LV) dysfunction. Hemodynamic load reversal during pre-HF (4

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weeks post-ACF; REV) results in rapid structural but delayed functional recovery. This study

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investigated myocyte and myofilament function in ACF and REV and tested the hypothesis that

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a myofilament Ca2+ sensitizer would improve VO-induced myofilament dysfunction in ACF and

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REV. Methods and Results. Following the initial Sham or ACF surgery in male Sprague-

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Dawley rats (200-240g) at Week 0, REV surgery and experiments were performed at Weeks 4

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and 8, respectively. In ACF, decreased LV function is accompanied by impaired sarcomeric

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shortening and force generation and decreased Ca2+ sensitivity, while in REV, impaired LV

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function is accompanied by decreased Ca2+ sensitivity. IV Levo elicited the best inotropic and

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lusitropic responses and was selected for chronic oral studies. Subsets of ACF and REV rats

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were given vehicle (Veh; water) or Levo (1 mg/kg) in drinking water from Weeks 4-8. Levo

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improved systolic (%FS, Ees, PRSW) and diastolic (tau, dP/dtmin) function in ACF and REV.

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Levo improved Ca2+ sensitivity without altering the amplitude and kinetics of the intracellular

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Ca2+ transient. In ACF-Levo, increased cMyBP-C Ser-273 and Ser-302 and cTnI Ser-23/24

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phosphorylation correlated with improved diastolic relaxation, while in REV-Levo, increased

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cMyBP-C Ser-273 phosphorylation and increased α-to-β-MHC correlated with improved

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diastolic relaxation. Conclusion. Levo improves LV function, and myofilament composition

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and regulatory protein phosphorylation likely play a key role in improving function.

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Keywords: (3-5 words or phrases)

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Myofilament dysfunction; Myofilament Ca2+ sensitization; levosimendan; myosin binding

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protein-C; troponin I

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Introduction

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Mitral regurgitation (MR) is the most and second most common valve lesion in the United States

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and Europe, respectively, affecting >2 million Americans [10,16]. MR’s pathophysiological

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consequences include chronic left ventricular (LV) hemodynamic volume overload (VO)

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followed by LV chamber dilation, progressive LV contractile dysfunction and heart failure (HF).

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Despite VO’s clinical importance, few models mimic the pathophysiological progression of

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chronic VO with and without hemodynamic load reduction. The aortocaval fistula (ACF) model

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of VO-HF in the rodent mimics increased hemodynamic preload observed in human disease

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irrespective of etiology. In this model, chronically increased LV preload leads to progressive LV

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pump failure, which is classified into three clinically relevant stages: pre-HF (4 weeks ACF;

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marked LV dilation, increased LV wall stress, mild LV dysfunction (~10% decrease in %

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fractional shortening (%FS)); no pulmonary congestion/edema), established HF (8 weeks ACF;

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severe LV dilation, significant LV systolic (25% decrease in %FS, ~50% decrease in EES and

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PRSW) and diastolic dysfunction (10-27% increase in Tau and dP/dtmin, respectively); no

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pulmonary congestion or edema) and end-stage HF (15-21 weeks ACF; LV pump failure with

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pulmonary congestion/edema) [17,19,34]. This model is commonly used to identify molecular

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and cellular mechanisms driving disease progression and to test novel therapeutic approaches for

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improving LV structure and function [13,21,40].

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We have previously characterized a technique to reverse (REV, close) the ACF [19]. In that

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report, REV during pre-HF (i.e. 4 weeks post-ACF) results in rapid structural recovery but

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delayed functional recovery [19]. While this mirrors human data where structure, but not

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function, returns post-surgically in 17% of MR patients [27], the mechanism underlying the

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delayed functional recovery remains elusive. Although the cellular mechanisms that control

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excitation-contraction coupling have been studied at end-stage HF [17], less is known about

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cellular dysfunction at earlier stages of HF. Specifically, myocyte and myofilament function has

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not previously been evaluated in ACF or REV and our model provides an attractive system for

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studying the physical and biochemical mechanics of VO following before and after surgical

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intervention.

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Additionally, this model is attractive because pharmacologic agents can be added in the presence

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or absence of hemodynamic load reversal. VO-HF therapeutic options generally target

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neurohormonal pathways by disrupting receptor-ligand interactions or modulating downstream

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signaling pathways (e.g. Ca2+-cAMP). Although these therapies successfully manage LV

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hypertrophy, their inotropic actions do not directly target impaired LV contractility, the central

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feature of systolic HF [14]. This can result in increased myocardial oxygen consumption and

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myocardial Ca2+ overload [30]. Newer therapeutics target myofilament activation without

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altering the Ca2+ transient and these myofilament Ca2+ sensitizers (e.g. levosimendan, Levo) and

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myosin activators (e.g. omecamtiv mecarbil, OM), are used to treat acute and chronic HF

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[5,20,37].

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The goal of the present study was to investigate the effects of volume overload and volume

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overload reversal on myocyte and myofilament function. Based on these results, we then tested

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the hypothesis that a myofilament Ca2+ sensitizer would improve VO-induced myofilament

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dysfunction in ACF and REV.

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Materials and methods

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ACF and REV Surgical Model

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Male Sprague-Dawley rats (200-240g; Harlan, Charles River) were housed in a temperature and

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humidity-controlled room using a 12h light/dark cycle with free access to standard rat chow and

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water. Studies, approved by the Institutional Animal Care and Use Committee, conformed to the

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NIH’s “Guide for the Care and Use of Laboratory Animals”.

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At Week 0, VO was induced under isoflurane anesthesia (2-2.5%) as described [17,19].

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Following abdominal incision, the abdominal aorta and caudal vena cava were exposed. Cranial

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and caudal to the fistula site, the adventitia was bluntly separated and 5-0 Ethilon® suture

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(Ethicon, Cincinnati, OH) was pre-placed for reversal. An 18g needle was used to create the

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ACF. The aortic puncture was sealed with cyanoacrylate glue, and the pre-placed suture was

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loosely tied taking care to not occlude blood flow. In a subset of rats, the fistula site was

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exposed and the pre-placed suture was ligated 4 weeks post-ACF (“REV”). Shunt patency and

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subsequent closure were confirmed by the presence or absence, respectively, of bright red

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arterial blood in the vena cava. The abdominal wall and skin were closed. Sham animals

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underwent a similar procedure except for suture pre-placement and aortic puncture.

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Buprenorphine (0.03 mg/kg SC) was given immediately post-operatively and every 12 hours for

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72 hours post-operatively as needed for pain.

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Echocardiography

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Transthoracic echocardiograms were performed biweekly (8.5-MHz; Xario, Toshiba Medical

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Systems, Tustin, CA) under isoflurane anesthesia (1.5-2%) [17,19]. Mid-wall parasternal short-

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axis M-mode images were obtained to assess chamber diameters in end-systole (LVESD) and

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end-diastole (LVEDD) and LV posterior wall thickness in systole (PWTs) and diastole (PWTd).

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These indices were calculated: % Fractional shortening (%FS) = (LVEDD-

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LVESD)/LVEDDx100; Dilation index = (2*PWTd)/LVEDD.

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Measurement of sarcomere shortening and Ca2+-transients in isolated LV myocytes

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Hearts were removed from anesthetized rats for LV myocyte isolation [17]. The heart, mounted

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on a Langendorff apparatus, was perfused with Tyrode’s solution supplemented with 10mM 2,3-

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butanedione monoxime (BDM), followed by perfusion buffer and then digestion with

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trypsin/Liberase-TH. LV myocytes were mechanically dispersed, filtered, and resuspended in

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increasing [CaCl2] to achieve [CaCl2]final=1mM. Isolated myocytes were incubated on laminin-

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coated chambers in plating media, then culture media for 1 hour each.

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Single myocyte sarcomere shortening (1 Hz field stimulation) was measured with a Myocam

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(IonOptix Corporation, Milton, MA) within 2-3 h of initial plating. These parameters were

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analyzed: sarcomere peak shortening normalized to resting sarcomere length (%PS,), maximal

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cell shortening (-dL/dt) and relengthening (+dL/dt) velocities, and time to 90% peak contraction

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and relaxation. To evaluate Ca2+, myocytes were loaded with Fura-2AM [17]; they were excited

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during field stimulation with light in an interleaved pattern at 340/12 nm and 380/12 nm and

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emission was collected at 510/40 nm. Data were expressed as peak Ca2+ amplitude normalized

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to resting diastolic Ca2+ (peak[Ca2+]i), maximum rates in rise of systolic Ca2+ and Ca2+ decay,

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and times to 90% peak Ca2+ release and Ca2+ reuptake. All measurements were analyzed using

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IonWizard data acquisition system (IonOptix).

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Myofilament pCa-Force

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Myocyte fragments were isolated from frozen LV tissue by mechanical dissociation and

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chemically permeabilized with 0.3% Triton-X100 and used as described [2,4,22]. Active force

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development was fit to a modified Hill equation that yields maximum force development, Hill

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coefficient (a measure of cooperativity), and pCa50 (–log[Ca2+] where 50% of maximum force is

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developed, a measure of myofilament Ca2+ sensitivity).

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Acute intravenous levosimendan, milrinone and omecamtiv mecarbil treatment

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To investigate the effects of three positive inotropes with varying mechanisms of action, 8 week

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ACF rats were infused with levosimendan (“IV Levo”; 2.8 μg/kg/min; LKT Laboratories, St

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Paul, MN), milrinone (9.9 μg/kg/min; LKT Laboratories) or omecamtiv mecarbil (OM; 11.7

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μg/kg/min; Selleck Chemicals, Houston, TX) for 30 minutes. Additional Sham and ACF rats

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were infused with vehicle (1% DMSO in 5% dextrose in water). %FS and mean arterial pressure

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(MAP) were measured before and after infusion, and tau and dP/dtmin (see below) were measured

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post-infusion.

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Chronic oral levosimendan treatment

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From Weeks 4-8 (Figure 1), ACF and REV rats were given vehicle (Veh; water) or

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levosimendan (“oral Levo”; L-enantiomer of ([4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-

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pyridazinyl)phenyl]-hydrazono)-propanedinitrile; LKT Laboratories, MN). Levo (0.0133

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mg/mL), prepared twice-weekly in drinking water, delivered a dose of ~1 mg/kg/day, which has

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improved LV function in previous rodent studies [6,24,25].

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Radiotelemetry

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Radiotelemetry catheters (PhysioTel PA-C40; Data Sciences International, St. Paul, MN) were

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implanted immediately following Sham or ACF surgery. Following ventral neck incision, the

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catheter was inserted into the right carotid artery to the aortic arch. The radiotransmitter was fed

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into a subcutaneous pocket positioned dorsally over the right shoulder, and the neck incision was

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closed. Blood pressure was recorded using Dataquest A.R.T (Data Sciences International)

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acquisition software. Final analysis averaged data from 24-hour periods.

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Dobutamine stress echocardiography

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Following baseline echo at Week 8, dobutamine (0.5 mg/kg IP) was given to a subset of ACF

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and REV rats given Veh or oral Levo. M-mode images were obtained 2.5-5 min post-injection;

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%FS at the peak increase was analyzed.

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Invasive LV hemodynamics

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LV hemodynamics were assessed during Week 8 with a PV catheter (1.9F, SciSense, London,

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ON) as described [17,19]. Rats were anesthetized with 3% isoflurane, intubated by tracheostomy,

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ventilated (SAR-830, CWE Inc, Ardmore, PA), and maintained under 1.75% isoflurane

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anesthesia. Baseline LV hemodynamic parameters were acquired; preload was varied by brief

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occlusion of the vena cava to obtain preload-recruitable stroke work (PRSW) and end-systolic

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elastance (Ees), the slope of the end-systolic pressure volume relationship (ESPVR). Data was

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acquired and analyzed using Labscribe2 software (iWORX, Dover, NH). Measures of LV

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function include stroke volume (SV), heart rate (HR), maximum and minimum dP/dt, end-

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systolic and end-diastolic volume (ESV and EDV), end-systolic and end-diastolic pressure (ESP

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and EDP), Ees, PRSW, and tau (Weiss correction). Analysis of covariance (ANCOVA)-adjusted

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marginal means of Ees and PRSW are presented and account for changes in the volume-axis

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intercept [8].

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Immunoblot analysis

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Immunoblots of LV tissue lysates were performed and analyzed as described [17,19] with

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antibodies against SERCA-2a (1:2,000, Thermo, Waltham, MA), phospholamban (PLB, 1:2,000,

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Millipore, Billerica, MA), phospho-PLB Ser16 (p-PLB, 1:2,000, Millipore), cTnI (1:2000, Cell

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Signaling, Beverly, MA), phospho-TnI Ser23/24 (1:1000, Cell Signaling), cMyBP-C (1:5000)

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[15], and phospho-cMyBP-C Ser-273, Ser-282 and Ser-302 (1:5000) [15].

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Quantitative real-time PCR analysis.

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RNA was isolated from LV tissue and amplified for α and β-MHC as described [17,19]. Data

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were analyzed for relative expression using the 2−ΔΔCt method, with ribosomal protein Rpl13a

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as the internal control and the average Sham value as a second normalizer.

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Statistical analysis

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Data are expressed as mean±SEM. Statistical analyses were performed using GraphPad Prism

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V6.0 or SPSS V19. One-way or two-way ANOVA or ANCOVA, followed by Bonferroni's post-

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hoc test, was used to measure differences between groups. p0.05) and the time to

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90% of peak relaxation (0.51±0.01 ms, 0.51±0.01 ms, 0.49±0.01 ms, respectively; p>0.05) were

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not different.

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ACF alters myocyte force generation and myofilament Ca2+ sensitivity without altering Ca2+

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transient kinetics

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To further investigate changes in excitation-contraction coupling, we next measured the

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amplitude and kinetics of the Ca2+ transient. Representative Ca2+ transients are depicted in

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Figure 2E. The mean peak amplitude of Ca2+ fluorescence was not different in Sham, ACF and

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REV myocytes (Figure 2F). Additionally, there were no differences in the peak rate of Ca2+

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release (Figure 2G), the time to 90% of peak [Ca2+]i, (0.03±0.00 ms, 0.03±0.00 ms, 0.03±0.00

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ms, respectively; p>0.05) or the time to remove 90% of [Ca2+]i towards baseline (0.54±0.01 ms,

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0.54±0.01 ms, 0.55±0.01 ms, respectively; p>0.05). Compared to Sham, the peak rate of Ca2+

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reuptake was decreased (less negative) in REV but unchanged in ACF (Figure 2H).

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Finally, we evaluated alterations in the expression of SERCA-2a and total and phosphorylated

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phospholamban (PLB) in LV tissue. Representative immunoblots are shown in Figure 3A and

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summary data is shown in Figures 3B-C. SERCA-2a was increased 2.5-fold in ACF and

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normalized in REV. Although these changes in SERCA-2a may be compensatory, there is poor

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correlation with the peak rate of Ca2+ reuptake or the peak rate of myocyte relengthening. This

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suggests that the changes in SERCA-2a may be unrelated to the pathophysiology of myocyte and

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myocardial dysfunction in ACF and REV. Total and phospho-PLB/PLB were not statistically

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different amongst Sham, ACF and REV.

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With the limited alterations in ACF or REV myocyte calcium transient or kinetics, we next

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measured myofilament Ca2+ sensitivity and force generation. Myofilament force generation at

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varying [Ca2+] was measured in skinned myocytes from frozen LV tissue. Compared with Sham,

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there was a rightward shift in the force-pCa relationship for ACF and REV (5.37±0.01 and

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5.40±0.02 vs. 5.47±0.02, respectively) indicating decreased Ca2+ sensitivity and 16% decrease in

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maximal force generation for ACF (Figure 3D). Maximal force generation was comparable in

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Sham and REV.

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Acute IV Levo and milrinone improve LV systolic and diastolic function

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The findings from the myocyte and myofilament studies demonstrate decreased myofilament

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Ca2+ sensitivity in ACF and REV without alterations in Ca2+ handling. Based on these findings,

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we next treated ACF rats with pharmacologic agents that improve myofilament function (i.e.

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milrinone, levosimendan or omecamtiv mecarbil) IV to determine their effects on systolic and

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diastolic function. Following 30 min infusion, %FS in ACF (32%) was increased by IV Levo

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(40%), milrinone (38%) and OM (37%); MAP remained unchanged in all groups (Figure 4A).

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Diastolic relaxation differed amongst groups (Figure 4B). Diastolic relaxation was normal in

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Sham and impaired in ACF-Veh. Diastolic relaxation as measured by tau and dP/dtmin was

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improved in ACF-Levo and ACF-Milrinone, but impaired in ACF-OM. Based on these results,

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Levo was selected for chronic oral treatment to determine whether IV Levo preserves/improves

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LV function in ACF and enhances functional recovery in REV (Figure 1).

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Chronic oral Levo does not significantly alter LV chamber morphology

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Compared with Sham at Weeks 6 and 8, LVEDD was increased in ACF-Veh and ACF-Levo and

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normalized in REV-Veh and REV-Levo (Figure 5, Table 1). At these timepoints, LVESD was

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increased to a greater degree in ACF-Veh and REV-Veh compared with ACF-Levo and REV-

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Levo. Since PWTd remained largely unchanged, the dilation index was significantly lower in

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ACF but normalized in REV, indicating eccentric hypertrophy and reverse remodeling,

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respectively (Table 1). EDV and SV were significantly increased in ACF, but reduced in REV

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(Table 1). In ACF-Veh and ACF-Levo, these increases were accompanied by increased heart

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and wet lung weights (Table 1), consistent with pathologic LV hypertrophy and pulmonary

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congestion/edema. By contrast, heart and lung weights normalized to Sham levels in REV-Veh

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and REV-Levo.

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Chronic oral Levo improves LV systolic and diastolic function without altering β-adrenergic

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responsiveness

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%FS was measured by serial echocardiography (Figure 5). At 2-4 weeks post-ACF, %FS was

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significantly lower in ACF compared to Sham. Oral Levo significantly improved %FS (8%

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increase compared with Week 4, p0.05) amongst all five groups: Sham (110±3 mmHg), ACF-

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Veh (103±2 mmHg), ACF-Levo (111±4 mmHg), REV-Veh (120±2 mmHg), and REV-Levo

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(116±3 mmHg).

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Chronic oral Levo improves LV myocyte sarcomere shortening kinetics without significantly

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altering peak shortening

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To evaluate the effects of oral Levo on peak sarcomere shortening, LV myocytes from Levo-

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treated rats were isolated at Week 8. Representative sarcomere shortening recordings are shown

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in Figure 8A. Peak sarcomere shortening and maximal sarcomere shortening velocity (-dL/dt) in

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ACF-Levo and REV-Levo was not different from ACF-Veh or REV-Veh, respectively, or Sham

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(Figures 8B-C). Maximal sarcomere relengthening velocity (+dL/dt) in ACF-Levo was not

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different from ACF-Veh or Sham, but increased in REV-Levo compared with Sham and no

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different from REV-Veh (Figure 8D). By contrast, compared to Sham, ACF-Veh, and REV-

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Veh, the time to 90% of peak contraction and relaxation was decreased in ACF-Levo and REV-

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Levo myocytes (Figures 8E-F).

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Chronic oral Levo improves myofilament Ca2+ sensitivity and maximal force generation without

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altering myocyte Ca2+ transient kinetics

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To further investigate changes in excitation-contraction coupling in Levo-treated rats, we next

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measured the amplitude and kinetics of the Ca2+ transient. Representative Ca2+ transients are

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depicted in Figure 9A. The mean peak amplitude of Ca2+ fluorescence was 30% decreased in

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ACF-Levo compared with ACF-Veh (p>0.0001) and 11% decreased in REV-Levo compared

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with REV-Veh (p>0.05; Figure 9B). In ACF-Levo, the peak rates of Ca2+ release and reuptake

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were not different from Sham or ACF-Veh (Figures 9C-D); however in REV-Levo, the peak rate

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of Ca2+ release was 37% greater than REV-Veh and the peak rate of Ca2+ reuptake was 29% and

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53% greater than Sham and Rev-Veh, respectively (Figures 9C-D). Finally, in ACF-Levo and

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REV-Levo, the time to 90% of peak [Ca2+]i and the time to remove 90% of [Ca2+]i towards

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baseline were not different from Sham, ACF-Veh or REV-Veh.

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Finally, we evaluated alterations in the expression of SERCA-2a and total and phosphorylated

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phospholamban (PLB) in LV tissue. Representative immunoblots are shown in Figure 9E and

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summary data is shown in Figures 9F-G. SERCA-2a was increased 1.6-fold in ACF-Levo, but

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comparable with ACF-Veh and normalized in REV-Levo. Total and phospho-PLB/PLB were

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not statistically different amongst the groups.

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We next measured myofilament force generation at varying [Ca2+] in skinned myocytes from

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frozen LV tissue (Figure 9H). Oral Levo caused the leftward shift in the force-pCa relationship

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expected for a myofilament Ca2+ sensitizer, and in ACF-Levo, there was a ~50% increase in

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maximal force generation compared with ACF-Veh.

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Chronic oral Levo’s positive lusitropy is associated with increased myofilament regulatory

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protein phosphorylation and increased α-to-β-MHC ratio

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Diastolic relaxation is controlled by changes in Ca2+ cycling, myofilament composition, Ca2+

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desensitization, and crossbridge cycling kinetics [1,7]. Coordinated phosphorylation of cMyBP-

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C and cTnI are central to these processes; therefore, we measured cMyBP-C and cTnI

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phosphorylation at key serine residues (cMyBP-C Ser-273, Ser-282 and Ser-302 and cTnI Ser-

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23/24). Total protein levels, though variable, were not different amongst groups (n=5-6; Figure

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10). In ACF-Levo cMyBP-C phosphorylation at Ser-273 (~2-fold) and Ser-302 (~3-fold), but not

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Ser-282 (not shown), were significantly increased compared to ACF-Veh (Fig 6a). Additionally

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in ACF-Levo, cTnI Ser-23/24 phosphorylation (~2 fold) was significantly increased (Figure

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10B). By comparison in REV-Levo, only cMyBP-C phosphorylation at Ser-273 was

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significantly increased (~2 fold).

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Because of the role of myofilament composition in cross-bridge cycling kinetics, we measured α-

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and β-MHC mRNA in LV tissue. As previously demonstrated in ACF-Veh and REV-Veh [19]

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there is a relative decrease and increase in α- and β-MHC mRNA, respectively (Figure 10C).

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While the α-to-β-MHC mRNA ratio remains depressed in ACF-Levo compared with Sham (4.9

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vs. 11.2, respectively), the α-to-β-MHC mRNA ratio is normalized in REV-Levo (10.2) and

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significantly improved relative to REV-Veh (5.6, p