incomplete recovery of myocyte contractile function...

Incomplete Recovery of Myocyte Contractile Function Despite Improvement of

Myocardial Architecture With Left Ventricular Assist Device Support

Ambardekar et al: Myocyte recovery after LVAD

Amrut V. Ambardekar MD; John S. Walker PhD; Lori A. Walker PhD; Joseph C.

Cleveland Jr. MD; Brian D. Lowes MD, PhD; Peter M. Buttrick MD

Divisions of Cardiology and Cardiothoracic Surgery

University of Colorado Denver, Aurora, CO

Correspondence to: Amrut V. Ambardekar, MD Division of Cardiology, University of Colorado Denver 12631 E. 17th Avenue, Room 7102, Campus Box B-130 Aurora, CO 80045 Email: [email protected] Telephone: 303-883-4278 Fax: 303-724-2094

Journal Subject Codes: [37] CV surgery: transplantation, ventricular assistance, cardiomyopathy; [105] Myocardial biology: Contractile function; [107] Myocardial biology: Biochemistry and metabolism; [110] Congestive Heart Failure

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Abstract

Background Unloading a failing heart with a left ventricular assist device (LVAD) can

improve ejection fraction (EF) and left ventricular (LV) size; however, recovery with

LVAD explantation is rare. We hypothesized that evaluation of myocyte contractility

and biochemistry at the sarcomere level before and after LVAD may explain organ level

changes.

Methods and Results Paired LV tissue samples were frozen from 8 patients with

nonischemic cardiomyopathy at LVAD implantation (Before LVAD) and prior to

transplant (After LVAD). These were compared to 8 nonfailing hearts. Isolated skinned

myocytes were purified, attached to a force transducer, and dimensions, maximal calcium

saturated force (Fmax), calcium sensitivity, and myofilament cooperativity were

assessed. Relative isoform abundance and phosphorylation levels of sarcomeric

contractile proteins were measured. With LVAD support, the unloaded EF improved

(10.0±1.0 to 25.6±11.0%, p=0.007), LV size decreased (LVIDd 7.6±1.2 to 4.9±1.4cm,

p<0.001), and myocyte dimensions decreased (cross-sectional area 1247±346 to

638±254 m2, p=0.001). Fmax improved after LVAD (3.6±0.9 to 7.3±1.8mN/mm2,

p<0.001), but was still lower than nonfailing (7.3±1.8 vs. 17.6±1.8mN/mm2, p<0.001).

An increase in troponin I (TnI) phosphorylation after LVAD was noted, but protein

kinase C phosphorylation of TnI decreased. Biochemical changes of other sarcomeric

proteins were not observed after LVAD.

Conclusions There is significant improvement in LV and myocyte size with LVAD,

but there is only partial recovery of EF and myocyte contractility. LVAD support was

only associated with biochemical changes in TnI. This suggests that alternate

mechanisms might contribute to contractile changes after LVAD and that additional

interventions may be needed to alter biochemical remodeling of the sarcomere to further

enhance myofilament and organ level recovery.

Key Words: mechanical circulatory support, remodeling heart failure, mechanical

unloading, heart failure

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ve isoform abundance and phosphorylation levels of sarcom

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=0 001) Fmax improved after LVAD (3 6±0 9 to 7 3±1 8mN

ve isoform abundance and phosphorylation levels of sarcom

ins were measured. With LVAD support, the unloaded EF im

6±11.0%, p=0.007), LV size decreased (LVIDd 7.6±1.2 to 4

myocyte dimensions decreased (cross-sectional area 1247±346

=0 001) Fmax improved after LVAD (3 6±0 9 to 7 3±1 8mN


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Abbreviations

LVAD=left ventricular assist device, EF=ejection fraction, LV=left ventricle,

LVIDd=left ventricular internal dimension end-diastole, TnI=troponin I, HF=heart

failure, MyBPC=myosin binding protein C, TnT=troponin T, Tm=tropomyosin, MLC-

2=regulatory myosin light chain, MLC-1=essential myosin light chain, PKA=protein

kinase A, PKC=protein kinase C, SD=standard deviation


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The left ventricular assist device (LVAD) is a well established therapy for patients

with end-stage heart failure (HF). The mechanical unloading of the left ventricle with the

LVAD and the subsequent restoration of cardiac output results in improvements in HF

symptoms, functional status, quality of life, and end-organ perfusion. In addition to these

systemic effects, some patients undergoing LVAD support demonstrate improved

function of the native left ventricle (LV), termed reverse remodeling. Such organ level

improvements include decreased LV chamber size, decreased LV mass, and improved

LV ejection fraction (EF)1 and have been accompanied by systemic effects such as

normalization of catecholamine levels,2 natriuretic peptides levels,3 and circulating

cytokines like TNF- .1,4

Some of these clinical markers of reverse remodeling at the organ level have also

been demonstrated at the cellular and molecular level. Previous studies have noted

reduction in myocyte hypertrophy5,6 as well as improvement in overall cardiac histology7

after LVAD support. Furthermore, in vitro studies have demonstrated improvements in

myocardial contractile properties, restoration of adrenergic receptor density and

responsiveness, and improved calcium handling with LVAD support.8-11

As both clinical and cellular evidence suggested the plausibility of reversing end-

stage disease, the concept arose that LVADs may be used as a bridge to recovery in HF

patients. Indeed, this was met with great enthusiasm after a single center’s report of the

successful explantation of LVADs in 11 of 15 patients with nonischemic cardiomyopathy

treated with a combination of unloading with a LVAD followed by conventional HF

medical therapy and the selective 2 adrenergic receptor agonist clenbuterol.12 However,

such high rates of recovery were not observed in the multi-institutional LVAD Working

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these clinical markers of reverse remodeling at the organ lev

ted at the cellular and molecular level. Previous studies have

ocyte hypertrophy5,6 as well as improvement in overall cardia


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Group study where only 6 of 67 patients (9%) underwent LVAD explantation for

recovery.1

At this time, the mechanisms of reverse remodeling remain poorly understood,

and it appears that restoring myocardial contractility may be more complex than what had

been initially considered. Therefore, we postulated that evaluation of myocyte

contractility and biochemistry at the most fundamental contractile level of the heart, the

sarcomere, before and after LVAD placement might reveal an explanation for this

phenomenon. Our first goal was to assess sarcomeric contractile properties by measuring

direct isometric forces on skinned isolated cell preparations. Second, since the cardiac

sarcomeric proteins myosin binding protein C (MyBPC), troponin T (TnT), troponin I

(TnI), tropomyosin (Tm), and regulatory myosin light chain (MLC-2) are known to effect

contractility,13 we assessed whether post-translational modifications and/or other

alterations in these proteins were affected with LVAD support.

Methods

Patient Selection and Tissue Acquisition

Paired LV tissue samples were collected and flash frozen from 8 patients with

end-stage nonischemic cardiomyopathy at the time of LVAD implantation (Before

LVAD) and prior to cardiac transplantation (After LVAD). Nonfailing control samples

were obtained from 8 donor hearts that were harvested for transplant but then unused for

non-cardiac reasons. The tissue obtained at the time of LVAD implantation consisted of

a core sample from the LV apex – at the in-flow cannula of the LVAD. The tissue

collected at the time of the cardiac transplant consisted of an analogously sized fragment

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sin (Tm), and regulatory myosin light chain (MLC-2) are kn

we assessed whether post-translational modifications and/or o

ese proteins were affected with LVAD support.


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excised from the explanted heart. All tissue samples were immediately flash frozen in

liquid nitrogen in the operating room (without the use of any cardioplegia solutions) and

transported to a -80 ºC freezer where they were stored until use. This storage method has

been shown to preserve sarcomeric contractile protein phosphorylation state and also

allow for the accurate assessment of the myocyte contractile parameters that are

described below.14-16 The ischemia time from removal of LV tissue from the patient to

freezing in liquid nitrogen for the Before LVAD samples was less than 10 seconds and

for the After LVAD and Nonfailing samples was less than 10 minutes. The Colorado

Multicenter Institutional Review Board approved the protocol for the collection, storage,

and analysis of human tissue.

Medical records were retrospectively reviewed by a trained physician to obtain

demographic and clinical data. Echocardiographic and hemodynamic data were obtained

from the medical record at the time closest to before LVAD implantation (to ensure

conditions reflecting the time the Before LVAD tissue was obtained) followed by the

time closest to cardiac transplantation (to ensure conditions reflecting the time the After

LVAD tissue was obtained). The data obtained from patients being supported with a

LVAD reflect the device settings that were clinically indicated at the time for the

patients, and represent the combined effects of native LV function as well as LVAD

related unloading.

Myocyte Contractility Measurements

Details of the myocyte isolation and experimentation protocols have been

previously described.14,17-19 Briefly, myocytes were purified from the frozen LV samples

ol for the collececccccctti

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records were retrospectively reviewed by a trained physician

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l record at the time closest to before LVAD implantation (to


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by mechanical homogenization and subsequently permeabilized with 0.3% Triton X-100.

The resulting suspension was placed on the stage of an inverted microscope, and in order

to ensure adequate tissue quality, myocyte fragments with an organized myofibril pattern

with clear sarcomeric striations were chosen for the contractility experiments.15 These

isolated skinned myocyte fragments were then attached to a force transducer and motor

and mechanical experiments were conducted. A representative photomicrograph is

shown in Figure 1. The length and width of the skinned myocyte fragment were

measured when it was attached to the force transducer in relaxing solution and a side

view mirror was used to measure the thickness. Cross-sectional area was calculated

using an elliptical approximation. The isolated myocytes were stored on ice and used

within 20 hours of isolation. All experiments were performed at 15 °C and since

sarcomere length can effect force measurements, the sarcomere length was set to 2.1 um

for all experiments.18

Myocytes were exposed in random order to six different activation solutions

containing varying concentrations of calcium (pCa 9.0 to 4.5) and the developed force for

each of the calcium concentrations was recorded as shown in Figure 2a. The developed

force at the maximal calcium concentration was measured before and after obtaining the

measurements of force at the other 6 calcium concentrations. If there was >15% decline

in force between the first and last force measurement at the maximal calcium

concentration, we considered this as evidence of cell fragment degradation and this data

was excluded from the analysis. Using these measurements, a force-pCa curve was

plotted (Figure 2b) and the Hill equation was fit to derive the following contractile

parameters: Fmax (the maximal calcium saturated developed force normalized to the

nal area was cccccccaalaaaaa

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of isolation. All experiments were performed at 15 °C and s

h can effect force measurements, the sarcomere length was se

nts.18


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cross-sectional area of the myocyte), pCa50 (the calcium concentration at which the force

is half maximal, a measure of calcium sensitivity), and Hill coefficient (the slope of the

calcium-force relation, an index of myofilament cooperative activation). In order to

minimize measurement variations for any single isolated myocyte, the individual patient

contractile data reflect the average of measurement recordings for 3 to 5 myocytes per

patient.

Sarcomeric Protein Phosphorylation Assessment

The remaining myocyte preparations not used for mechanical study were acetone

precipitated to clamp their phosphorylation state and homogenized in 8M urea, 2.5M

thiourea, 4% CHAPS, 10 mM EDTA, and a mixture of protease and phosphatase

inhibitors as previously described.20 Protein concentration of these samples was

measured using a BCA Protein Assay Kit (Thermo Scientific).

Phosphorylation levels of the cardiac sarcomeric proteins were determined by

separating the proteins with 12% 1D-SDS-PAGE and fixing and staining with a

phosphoprotein stain (ProQ Diamond Phosphoprotein Gel Stain, Invitrogen). After

imaging, the same gels were rinsed and stained with a total protein stain (BioSafe

Coomassie Blue, BioRad). All gels were imaged using a Typhoon 9410 Gel Imager (GE

Lifesciences) and protein optical densities were measured using ImageJ (version 1.42

NIH). In order to adjust for subtle differences in protein loading, the phosphorylation

levels were calculated by dividing the optical density of the each sarcomeric protein on

the ProQ diamond gel by the optical density of the same protein on the Coomassie gel.

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HAPS, 10 mM EDTA, and a mixture of protease and phospha

viously described.20 Protein concentration of these samples w

a BCA Protein Assay Kit (Thermo Scientific).


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In the case of MyBPC, the total protein stain optical density merges with the abundant

protein myosin, so the phosphorylation level of MyBPC on the phosphoprotein stain was

normalized to essential myosin light chain (MLC-1) as prior studies have reported.20

Additional Assessment of Sarcomeric Proteins

Site-specific phosphorylation of troponin I was assessed by Western blots.

Proteins were separated with 12% 1D-SDS-PAGE as above and transferred to PVDF

membranes. After the membranes were blocked in 5% BSA and rinsed with TBST, they

were incubated overnight at 4 °C with a phosphospecific primary antibody to TnI

phosphorylated at the putative protein kinase A (PKA) site, Serine 22,23 (Cell Signaling

1:1000) or to troponin I phosphorylated at the putative protein kinase C (PKC) site Serine

43 using an epitope specific phosphoserine antibody (Abcam 1:1000) as previously

described.20 The blots were then washed and incubated with secondary antibody (anti-

mouse from Sigma, 1:10000) for one hour at room temperature, washed, and visualized

using enhanced chemiluminescence. Membranes were stripped and subsequently

incubated in primary total cardiac TnI antibody (Fitzgerald, Inc 1:2500). Site specific

phosphorylation was calculated by dividing the optical density of the phosphospecific

antibody blot by the density of the total protein blot.

Changes in myosin heavy chain isoforms were assessed as previously described20

by separating proteins using modified 6% 1D-SDS-PAGE and subsequent staining with

BioSafe Coomassie Blue total protein stain. MLC-1 and MLC-2 phosphorylation and

isoforms changes were assessed using 2D-SDS-PAGE21 and percent phosphorylated

MLC-2 and percent atrial isoform of MLC-1 were calculated.

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ponin I phosphorylated at the putative protein kinase C (PKC

ope specific phosphoserine antibody (Abff cam 1:1000) as prev

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Changes in TnT isoform expression were assessed using by modified Western

blots as previously described.22 The primary cardiac TnT isoform antibody used was Ab-

1 (Clone 13-11, Thermo scientific 1:1000).

Statistical Analysis

Results are expressed as mean ± standard deviation (SD). The paired t-test was

used to compare differences between before and after LVAD implantation. The unpaired

t-test was used to compare differences between before LVAD implantation failing and

nonfailing and between after LVAD implantation failing and nonfailing groups.

Statistical significance was defined as a two-tailed P-value of less than 0.05. The R

version 2.9.1 (Vienna, Austria) statistical program was used for all analyses.

Results

Patient Characteristics

The baseline characteristics of patients with end-stage nonischemic

cardiomyopathy requiring LVAD as a bridge to transplant are provided in Table 1. All

patients were inotrope dependent with NYHA class IV HF at the time of LVAD

implantation, and 7 of 8 patients required mechanical support with an intra-aortic balloon

pump. The mean duration for the diagnosis of HF at the time of LVAD implantation was

59±22 months. Both first generation pulsatile displacement LVADs (HeartMate XVE,

Thoratec, Pleasanton, CA) and second generation continuous axial flow LVADs

(HeartMate II, Thoratec, Pleasanton, CA) were utilized. The mean duration of LVAD

support was 143±41 days. LVAD support resulted in significant reductions in LV

nonfailing grrououooooo

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iienna, Austria) statistical program was used for all analyses.


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echocardiographic dimensions, improvement in EF, and normalization of several

hemodynamic measures as summarized in Table 2.

The 8 nonfailing patients (3 male and 5 female) were organ donors whose hearts

were not utilized for transplant for non-cardiac reasons. The mean age of this group was

48 ± 8 years, the mean EF 70.6 ± 5.4%, and none of these patients had any prior cardiac

history. Seven (87.5%) of these patients were on adrenergic vasopressor agents during

the period of brain death and as organs were being harvested.

Myocyte Size and Contractility Parameters

Significant differences were noted for all myocyte fragment dimensions as

summarized in Table 3. Myocytes obtained from patients before LVAD implant were

significantly larger than myocytes from patients after LVAD implantation. However, the

myocytes from patients after LVAD implantation were still larger than the myocytes

obtained from nonfailing patients. Fmax doubled with LVAD support (3.6 ± 0.9 vs. 7.3 ±

1.8 mN/mm2, p<0.001), but was still less than half of nonfailing subjects (7.3 ± 1.8 vs.

17.6 ± 1.8 mN/mm2, p<0.001) as described in Table 4. The pCa50 was unchanged in all

groups and the Hill coefficient was reduced in both LVAD groups relative to controls.

Sarcomeric Proteins and Phosphorylation Levels

Total TnI phosphorylation increased with LVAD support (12.8 ± 4.1 vs. 21.6 ±

9.4 OD units, p=0.030) (Table 5 and Figure 3). While there were no significant

differences noted in the Serine 22,23 site specific phosphorylation of TnI, Serine 43 site

specific phosphorylation of TnI decreased after LVAD (7.5 ± 2.2 vs. 5.2 ± 1.9 OD units,

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Table 3. Myocytes obtained from patients before LVAD imp

ger than myocytes from patients after LVAD implantation. H

patients after LVAD implantation were still larger than the m


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p=0.044) (Figure 4). Total phosphorylation levels of MyBPC were higher in both failing

groups compared with nonfailing donors (Table 5). Phosphorylation levels of TnT, Tm,

and MLC-2 were not significantly different before and after LVAD (Table 5, and Figure

3).

There were no changes noted on 2D-SDS-PAGE in the percent phosphorylation

of MLC-2 before and after LVAD (43±3 vs. 41±2%, p=0.341) and in the percent atrial

isoform of MLC-1 before and after LVAD (23±5 vs. 21±7%, p=0.711). Furthermore, no

differences were noted in myosin heavy chain isoforms before and after LVAD with only

the -isoform being expressed in both groups of patients (Figure 5). Finally there was no

evidence of altered TnT isoform expression in the before LVAD, after LVAD, and

nonfailing groups. Total TnT content was unchanged between these groups.

Discussion

The main finding of this study is that in isolated skinned myocytes, there is a

marked reduction in maximum developed force in patients with end-stage nonischemic

cardiomyopathy compared to nonfailing donors. This marked reduction in force is only

partially improved with LVAD support. Furthermore, there is strong evidence that

structural changes accompany these changes in force at the organ level (reduction in LV

size) as well as at the cellular level (reduction of all myocyte dimensions) and these

correlate with functional changes at both the organ level (improvement in EF) and

cellular level (improvement in maximum force). Despite these significant changes in

structural and mechanical parameters, unloading of the LV with LVAD support did not

gure 5). Finallllllly y yyyyy

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change the biochemical properties of the sarcomere other than a change in TnI

phosphorylation.

Our findings confirm that contractile dysfunction in human heart failure at least in

part resides at the level of the cardiac myofilament. This is independent of cell loss or

changes in the cardiac interstitium and certainly argues that therapies to treat heart failure

need to acknowledge sarcomeric function. To place these changes in context, a prior

report using a similar experimental protocol in human samples reported ~30% reductions

in myocyte Fmax in diabetics with preserved EF compared to control patients (14.6±1.7

vs. 20.6±3.7 mN/mm2, p<0.05).14 The Fmax of the failing groups before and after LVAD

from the current study are more dramatic (~60%) than seen in this prior report, consistent

with the severe reductions in contractile function at the organ level as assessed by EF.

There are a number of cellular and molecular changes in the heart that occur with

LVAD support and these encompass the most basic aspects of genetic regulation and

involve a complex, interconnected cascade of changes.23 While there have been two

prior studies that have assessed for changes in myocyte contractility with LVAD, this is

the first study to directly measure isometric force of skinned myocytes from paired

samples before and after LVAD support. The two prior studies (one of which used paired

pre/post LVAD samples) involved patients with both ischemic and

nonischemic/idiopathic cardiomyopathies. Using in vitro motility and unloaded cell

shortening assays, the authors reported improvements in contractility in myocytes

obtained from patients with HF vs. patients with HF supported by LVAD.8,9 Our

assessment of myocyte contractility also differs from the two prior reports in that we

limited our analysis to only those patients with nonischemic cardiomyopathy (a global

roups before ananananannndd

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pathologic process that involves the entire LV) as opposed to ischemic cardiomyopathy

(a regional pathologic process based on areas of infarct/ischemia from coronary artery

disease). The prior reports included data from patients with ischemic cardiomyopathy,

and the before LVAD tissue (from the LV apex) would be inherently different than the

after LVAD tissue depending on the regions of LV infarction making comparisons

difficult. The results of our study confirm that among patients with nonischemic

cardiomyopathy, improvement in contractility at the sarcomeric level is possible with

LVAD support; however normalization of forces to levels seen in nonfailing subjects

does not occur using standard LVAD protocols. This is concordant with organ level

assessment of contractility where EF improved from 10.0 to 25.6% with LVAD support;

which is still half that seen in a nonfailing control hearts. This may in part explain

clinically why the majority of LVADs cannot be successfully explanted.

The cellular mechanisms by which contractility is depressed in these HF samples

and why there is only partial reversal with mechanical unloading is not unequivocally

answered in this study, although there is suggestive evidence that points towards both

structural and biochemical mechanisms. The fact that the Hill coefficient, a marker of

protein cooperativity, was reduced in both HF groups, coupled with the changes in

MyBPC phosphorylation suggest that functional interactions between actin and myosin

might have been compromised in both HF groups.24 This hypothesis is supported by the

cell morphology data which suggest that sarcomere cross-bridge (and perhaps lattice)

spacing is tightly coupled with contractile performance both in the two HF groups and

also in the donor heart specimens, a finding which reflects previous studies

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ular mechanisms by which contractility is depressed in the e

f that seen in a nonfailing control hearts. This may in part ex

he majority of LVADs cannot be successfully explanted.

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demonstrating that contractile parameters in isolated muscle were influenced in a step-

wise fashion by osmotic determinants that influenced lattice architecture.25,26

The mechanism for the partial recovery of contractile function following LVAD

support might be in part reflective of contractile protein phosphorylation changes. Total

TnI phosphorylation was lower before LVAD support compared to after LVAD and

nonfailing samples which may be due to down-regulation of -adrenergic receptors in

inotrope dependent patients with end-stage HF. The increase in TnI phosphorylation

after LVAD might be reflective of normalization of -adrenergic receptor function.

Despite this, there was an increase in TnI phosphorylation at serine 43—a putative PKC

site—in the pre LVAD samples which decreased following LVAD support. This finding

is consistent with a substantial literature that suggests that heart failure, both in humans

and in experimental animals, is associated with an increase in PKC activation,27,28 and

that PKC dependent phosphorylation of TnI is associated with depressed myofilament

force development.29-31 The finding also provides a plausible explanation as to why

phosphatase treatment of myofilament preparations from HF patients improves

contractile performance despite overall low levels of overall contractile protein

phosphorylation.9 The fact that there was no change seen in TnI phosphorylation at the

putative PKA site, serine 22,23 despite postulated changes in adrenergic receptor activity

before and after LVAD, may reflect the somewhat promiscuous nature of this site which

can be targeted both by PKA and PKC,29 the former likely increasing after LVAD and the

later declining.

Not seen in this study were changes in phosphorylation of other contractile

proteins or isoform shifts before and after LVAD support. In particular, and in contrast

serine 43 a pupuppppp

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h a substantial literature that suggests that heart failure, both

ntal animals, is associated with an increase in PKC activation

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ntal animals, is associated with an increase in PKC activation

dent phosphorylation of TnI is associated with depressed my


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to studies in experimental animals, we did not see a shift at the protein level in the /

myosin heavy chain isoform ratio or in troponin T isoform distribution. This is

particularly important to note as mechanical unloading of the normal LV in experimental

animals does result in atrophy associated with a recapitulation of the fetal gene program32

whereas in this study, unloading was associated with a normalization of ventricular size

without isoform changes.

Limitations

We acknowledge a number of limitations to this study. The overall number of

patients in the analysis is small and all were from a single center’s LVAD program.

There were no differences in the results between patients supported with pulsatile-flow

HeartMate XVE pumps versus continuous-flow HeartMate II pumps, although our small

numbers preclude accurate subgroup analysis to definitively test for differences in the

type of mechanical unloading. In addition, the echocardiographic measures of LV size

and EF during LVAD support were obtained from retrospective review and were thus

obtained on the LVAD settings that were clinically indicated for the patient. While these

assessments do partially reflect the influence of the LVAD, some component of native

LV function also contributes as on pump measures of LV size and EF are often used in

the initial screening assessments to determine whether patients should have further

testing to assess for recovery.33,34 Myocyte dimensions were measured from the isolated

skinned myocyte fragments that were used for mechanical force measurements rather

than a specific histological stain of intact tissue. Despite this different methodology, the

cross-sectional areas obtained in this study were very similar to those reported recently

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ifferences in the results between patients supported with puls

pumps versus continuous-flow HeartMate II pumps, althoug

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using such traditional histological techniques6 and the overall trends are well in line with

other published reports.5

The nonfailing tissue was obtained from organ donors, and the majority of these

patients were receiving adrenergic agents prior to and during harvest, so adrenergic

mediated pathways were likely activated in these patients. Furthermore, concomitant

medical therapy during LVAD support, such as -blockade, varied and may have

influenced some of the sarcomeric mechanical properties as has been previously

demonstrated.35 In addition, while we believe the comparison between LVAD (before

and after) and nonfailing donor hearts are illuminating, it is worth noting that the tissue

procurement strategies were by necessity somewhat different in that the nonfailing and

after LVAD hearts were excised en bloc whereas the before LVAD specimens were taken

from small tissue cores obtained from beating hearts. Finally, the before and after LVAD

samples were obtained in the operating room after the patient was induced with general

anesthesia and placed on cardiopulmonary bypass. These procedures may have affected

the sarcomeric protein phosphorylation background. Despite these limitations, the

current study remains the most comprehensive examination of the effects of LVAD

support on the sarcomere and thus merits attention.

Conclusion

There is significant improvement in LV and myocyte size with LVAD support,

but there is only partial recovery of EF and myocyte contractility. The architectural

changes of LVAD support were associated with a limited spectrum of biochemical

changes which suggest that both lattice spacing and PKC activation might contribute to

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b w

rts were excised en bloc whereas the before LVAD specimen

e cores obtained from beating hearts. Finally, the before and

btained in the operating room after the patient was induced w


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contractile dysfunction. These data further suggest that additional interventions beyond

mechanical unloading may be needed to alter biochemical remodeling of the sarcomere to

further enhance myofilament and organ level recovery.


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Sources of Funding

Dr. Ambardekar is supported by a Research Fellowship Award from the Heart Failure

Society of America (St. Paul, MN). Additional work on this review was supported by

grants from the National Institutes of Health (HL077195 and HL101435).

Disclosures

None.


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Table 1. Baseline Patient Characteristics

Patient Characteristics (n=8) Mean age (years) 40.5 ± 11.5 Male gender 7 (88%) Caucasian 4 (50%) African American 4 (50%) Mean duration of HF prior to LVAD (months) 56 ± 22 NYHA Class IV 8 (100%) Mean duration of LVAD support (days) 143 ± 41 HeartMate XVE LVAD* 5 (62%) HeartMate II LVAD* 3 (39%)

Medical Therapy or Interventions Before LVAD Intravenous inotropic agent 8 (100%) Intravenous vasodilator 5 (62%) Beta-blocker 0 (0%) ACE inhibitor/ARB 2 (25%) Aldosterone antagonist 7 (88%) Diuretic 8 (100%) Implantable cardioverter-defibrillator 4 (50%) Cardiac resynchronization therapy 2 (25%) Intraaortic balloon pump 7 (88%)

Medical Therapy during LVAD Support Intravenous inotropic agent 0 (0%) -blocker 6 (75%) ACE inhibitor/ARB 7 (88%) Aldosterone antagonist 7 (88%) Diuretic 6 (75%)

HF=Heart Failure, LVAD=left ventricular assist device, NYHA=New York Heart Association, ACE=angiotensin-converting enzyme, ARB=angiotensin II receptor blocker *Thoratec, Pleasanton, CA

(6(6(6(6(6(6(62%2%2%2%2%2%2%) ) ) ) ) ))0 (00%)%)%)%)%)%)%)

(25%5%%%%%%)))))))( )

rcl

( )tagonist 7 (88%)

8 (100%) rdioverter-defibrillator 4 (50%)

chronization therapy 2 (25%)loon pump 7 (88%)


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Table 2. Echocardiographic and Hemodynamic Parameters Before and After LVAD Support

Before LVAD

After LVAD

P-value

Ejection fraction (%) 10.0 ± 1 25.6 ± 11 0.007 Left ventricular end-systolic internal dimension (cm)

6.8 ± 1.2 4.1 ± 1.3 <0.001

Left ventricular end-diastolic internal dimension (cm)

7.6 ± 1.2 4.9 ± 1.4 <0.001

Heart rate (beats/min) 96 ± 27 79 ± 14 0.13 Mean arterial pressure (mmHg) 72 ± 6 90 ± 12 0.016 Right atrial pressure (mmHg) 16.8 ± 6.9 1.3 ± 0.6 <0.001 Mean pulmonary artery pressure (mmHg)

40 ± 8 19 ± 6 0.006

Pulmonary capillary-wedge pressure (mmHg)

27 ± 7 5 ± 3 <0.001

Cardiac output (liters/min) 2.9 ± 0.8 4.7 ± 0.8 0.024 Cardiac index (liters/min/m2) 1.5 ± 0.4 2.5 ± 0.7 0.16

55 ± ± 3 33333

4 7777777 ±±±±±±± 0000000 8888888((liters/min/m2) 1.5 ± 0.4 2.5 ± 0.7


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Table 3. Isolated Skinned Myocyte Fragment Morphometric Data

Before LVAD After LVAD Nonfailing Length ( m) 105 ± 21 86 ± 14* 75 ± 10† Width ( m) 41 ± 6 33 ± 7* 23 ± 3† Thickness ( m) 37 ± 6 24 ± 5* 17 ± 4† Cross-sectional Area ( m2)

1247 ± 346 638 ± 254* 330 ± 111†

Volume ( m3) 138660 ± 63460 56923 ± 29858* 25435 ± 10803†

*P-value <0.05 for comparison between Before LVAD and After LVAD †P-value <0.05 for comparisons between Nonfailing vs. Before LVAD and for Nonfailing vs. After LVAD.


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Table 4. Mean contractile parameters among control patients and patients with nonischemic cardiomyopathy before and after LVAD implantation.

Before LVAD

After LVAD

P-value* Nonfailing

Fmax (mN/mm2) 3.6 ± 0.9 7.3 ± 1.8 <0.001 17.6 ± 1.8† pCa50 5.89 ± 0.08 6.03 ± 0.28 0.257 5.85 ± 0.14 Hill coefficient 1.64 ± 0.28 1.55 ± 0.45 0.579 2.30 ± 0.81‡

The Fmax is the maximum calcium-saturated developed force normalized to the cross-sectional area of the myocytes, pCa50 is the calcium concentration at which the force is half maximal and represents a measure of myofilament calcium sensitivity, Hill coefficient is the slope of the calcium-force relation and is an index of myofilament cooperative activation. *P-value for comparison between Before LVAD and After LVAD †P<0.001 for comparisons of Fmax between Before LVAD vs. Nonfailing and After LVAD vs. Nonfailing ‡P<0.05 for comparisons of Hill coefficient between Before LVAD vs. Nonfailing and After LVAD vs. Nonfailing

VAD ss. NoNoNoNoNoNoNonfnfnfnfnfnfnfaiaiaiaiaiaiailililililililingngngngngngng a aaaaaannnnnnn

m risons of Hill coefficient between Before LVAD vs. Nonfmparisons of Hill coefficient between Before LVAD vs. NonfNonfailing


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Table 5. Phosphorylation levels of sarcomeric proteins in patients with nonischemic cardiomyopathy before and after LVAD implantation.

Before LVAD

After LVAD

P-value* Nonfailing

Myosin Binding Protein C

6.1 ± 1.2 6.7 ± 1.0 0.328 4.6 ± 0.6†

Troponin T 19.2 ± 3.0 18.0 ± 3.8 0.562 20.9 ± 7.0 Troponin I 12.8 ± 4.1 21.6 ± 9.4 0.030 13.2 ± 1.9 Tropomyosin 7.9 ± 2.6 8.5 ± 2.9 0.431 8.0 ± 1.9 Regulatory Myosin Light Chain

0.5 ± 0.2 0.7 ± 0.3 0.109 0.6 ± 0.2

Serine 22,23 phosphorylated Troponin I

3.7 ± 2.3 3.4 ± 1.5 0.747 2.8 ± 0.6

Serine 43 phosphorylated Troponin I

7.5 ± 2.2 5.2 ± 1.9 0.044 12.0 ± 1.0‡

Phosphorylation levels expressed in arbitrary optical density units. *P-value for comparison between Before LVAD and After LVAD †P<0.01 for comparisons of Myosin Binding Protein C between Before LVAD vs. Nonfailing and After LVAD vs. Nonfailing ‡P<0.001 for comparisons of Serine 43 phosphorylated Troponin I between Before LVAD vs. Nonfailing and After LVAD vs. Nonfailing

1212.0.0 ± 1 1.0.0.0.0.0.0.0‡‡

mm AA

i f S i 43 h h l t d T i I b t B

levels expressed in arbitrary optical density units.

mparison between Before LVAD and After LVAD mparisons of Myosin Binding Protein C between Before LVAAfter LVAD vs. Nonfailing

i f S i 43 h h l t d T i I b t B


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Figure Legends

Figure 1. Photomicrograph example of an isolated skinned myocyte fragment attached to

the force transducer and motor in relaxing solution for mechanical contractile

experiments. The visible sarcomere striations were set at 2.1 m for all myocyte

experiments.

Figure 2.

(a) Representative tracing of force measurements from the experimental protocol. After

the myocyte had achieved a steady force in the calcium containing activation solution, it

was rapidly step shortened to break cross-bridges (top tracing) and the maximum force

for the particular activation solution was then recorded (bottom tracing).

(b) Representative plot of the force-calcium relation. Force is shown plotted against the

inverse log of the calcium concentration (pCa). The Fmax (the maximal calcium

saturated developed force normalized to the cross-sectional area of the myocyte), pCa50

(the calcium concentration at which the force is half maximal, a measure of calcium

sensitivity), and Hill coefficient (the slope of the calcium-force relation, an index of

myofilament cooperative activation) for each myocyte were derived from these force-pCa

curves.

Figure 3. 12% 1D-SDS-PAGE images stained with a total phosphoprotein stain (ProQ

Diamond Phosphoprotein Gel Stain) in panel A and a total protein stain (BioSafe

xperimental pprororororororot

ainingngngngngngng acacacacacacactititititititivavavavavavavatititititititionooooo

m

r

shortened to break cross-bridges (top tracing) and the maxim

r activation solution was then recorded (bottom tracing).


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Coomassie Blue) in panel B demonstrate the increase in total TnI phosphorylation with

LVAD support and higher MyBPC phosphorylation in both failing samples compared

with nonfailing donors. Sarcomeric protein phosphorylation levels were calculated by

dividing the optical density of the each sarcomeric protein on the ProQ diamond gel by

the optical density of the same protein on the Coomassie gel. MyBPC was normalized to

MLC-1 as noted in the text.

NF=nonfailing, MyBPC=myosin binding protein C, TnT=troponin T, Tm=tropomyosin,

TnI=troponin I, MLC-1=myosin light chain 1, MLC-2=myosin light chain 2

Figure 4. Western blots of phosphorylated troponin I (TnI) demonstrate while there were

no significant differences in Serine 22,23 site specific phosphorylation of TnI, Serine 43

site specific phosphorylation of TnI decreased after LVAD. Samples were separated on

12.5% SDS-PAGE and probed with antibodies against phosphoserine 22/23,

phosphoserine 43, and cardiac TnI (for total protein). NF=nonfailing.

Figure 5. 6% 1D-SDS-PAGE separation of and myosin before and after LVAD

demonstrates that only the -isoform is expressed in both groups. The last lane is a

mixture of 50% nonfailing human with 50% nonfailing mouse and illustrates adequate

separation of and myosin isoforms.

in light chain n 22 22222

l

f n

s e

rn blots of phosphorylated troponin I (TnI) demonstrate whil

fferences in Serine 22,23 site specific phosphorylation of Tn

sphorylation of TnI decreased after LVAD. Samples were se


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and Peter M. ButtrickAmrut V. Ambardekar, John S. Walker, Lori A. Walker, Joseph C. Cleveland, Jr., Brian D. Lowes

Architecture With Left Ventricular Assist Device SupportIncomplete Recovery of Myocyte Contractile Function Despite Improvement of Myocardial

Print ISSN: 1941-3289. Online ISSN: 1941-3297 Copyright © 2011 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation: Heart Failure published online May 3, 2011;Circ Heart Fail.

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