Cardiomyocyte Cell

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Cardiomyocyte cell cycle activation improves cardiac function

after myocardial infarction

Rutger J. Hassink a,* , Kishore B. Pasumarthi b , Hidehiro Nakajima b , Michael Rubart b , Mark H.Soonpaa b , Aart Brutel de la Rivire c , Pieter A. Doevendans a , and Loren J. Field b

a Department of Cardiology, University Medical Center, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands b The Wells Center for Pediatric Research and Krannert Institute of Cardiology, IndianaUniversity School of Medicine, Indianapolis, IN 46202-5225, USA c Department of cardio-thoracic surgery,University Medical Center, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands

IntroductionCardiomyocyte death is a common end-point in many forms of cardiovascular disease. It isgenerally agreed that cardiomyocytes in the adult mammalian heart exhibit some capacity tore-enter the cell cycle. 1 In addition, recent studies suggested that adult-derived stem cells mightcontribute to cardiomyocyte renewal in injured hearts. 2, 3 However, it is very clear that theseintrinsic processes are of insufficient magnitude to restore cardiac function after myocardialinfarction. A number of approaches have been explored to increase cardiomyocyte number ininjured hearts, with the hope that this would promote functional recovery. These include directtransplantation of cardiomyocytes or myogenic stem cells, 4, 5 treatment with cytokines tomobilize endogenous stem cells, 6 and cardiomyocyte cell cycle activation. 7, 8

Commitment to a new round of cell division requires transit through the restriction point. 9,10

Restriction point transit is regulated by the activity of the cyclin-dependent kinases CDK2and CDK4, and their obligate co-factors, the D-type cyclins. CycD/CDK activity in turn ispositively regulated by growth factors and negatively regulated by members of the CIP andKIP CDK inhibitor families. Previous studies generated transgenic mice expressing D-typecyclins under the transcriptional regulation of the alpha-cardiac myosin heavy chain (MHC)promoter in an effort to promote cardiomyocyte cell cycle progression. 11, 12 Targetedexpression of cyclin D1, D2 or D3 resulted in increased base line levels of cardiomyocyte DNAsynthesis in the adult myocardium. Cardiomyocyte DNA synthesis persisted followingmyocardial injury in the cyclin D2 transgenic mice (designated MHC-cycD2 mice), but not inthe cyclin D1 and D3 mice. Sustained cell cycle activity in MHC-cycD2 mice was accompaniedby an increase in cardiomyocyte cell number and concomitant reduction of infarct size. 12

Although this previous study demonstrated a progressive improvement in cardiac architecture

post-myocardial infarction, it was not clear whether this was associated with the appearanceof functional cardiomyocytes and a concomitant improvement in cardiac function. In this study,the impact of cardiomyocyte cell cycle activity on cardiac function was examined followingmyocardial injury. MHC-cycD2 mice and their non-transgenic siblings were subjected topermanent coronary artery occlusion. Cell cycle activity resulted in the accumulation of newlyformed myocardium in the MHC-cycD2 transgenic mice. Intracellular calcium transient

*Corresponding author: Tel: ++ 31 73 6992000; fax: ++ 31 73 6992763. E-mail address: [email protected] .Conflict of Interest: none declared.

NIH Public AccessAuthor ManuscriptCardiovasc Res . Author manuscript; available in PMC 2009 April 1.

Published in final edited form as:Cardiovasc Res . 2008 April 1; 78(1): 1825. doi:10.1093/cvr/cvm101.N I H

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imaging indicated that the newly formed myocardium was functionally coupled to the remotemyocardium. Moreover, intra-ventricular pressure-volume measurements revealed a positivecorrelation between the presence of newly formed myocardium and improved cardiac functionin the MHC-cycD2 transgenic mice. In contrast, no improvement in cardiac structure orfunction was observed in the non-transgenic siblings. These findings support the notion thatcardiomyocyte cell cycle activation can restore function in injured hearts.

MethodsTransgenic mice

The generation of the MHC-cycD2 transgenic line was described previously. 12 These animalsexpressed a mouse cyclin D2 cDNA under the transcriptional regulation of the mouse -cardiacmyosin heavy chain (MHC) promoter. Mice were maintained in a DBA/2J inbred background.Male mice were used for all studies. The investigation conforms with the Guide for the Careand Use of Laboratory Animals published by the US National Institutes of Health (NIHPublication No. 85-23, revised 1996).

MI model

MI was induced by ligation of the left coronary artery as described. 12, 13 Briefly, the animals

were intubated and ventilated with 2% isoflurane and supplemental oxygen. Via leftthoracotomy the left coronary artery was ligated at the inferior border of the left auricle. Theintercostal space and the skin incision were then closed with interrupted sutures, theendotracheal tube was removed, and the animal placed on a 37 Celsius heating pad (ColeParmer, Vernon Hills IL) under a 100% oxygen cover for 24 hours post surgery. Sham-operatedanimals underwent the same procedure without ligation of the coronary artery.

Cardiac function analysis

Pressure-volume measurements were obtained as described before. 14,15 At 7, 60 or 180 dayspost-MI or sham-operation, mice were anesthetized with 2% isoflurane supplemented with100% oxygen, intubated with an endotracheal tube and ventilated (Minivent 845; HugoSachsElektronik, March-Hugstetten, Germany) at 125 cycles/min and a tidal volume of 67 l/g.Mice were placed in supine position under a dissection microscope and connected to a feedback heating-lamp via a rectal temperature sensor for maintenance of stable body temperature at 37Celsius. A precalibrated four-electrode 1.4F pressure-volume (P-V) catheter (Model SPR-839;Millar Instruments, Houston, TX) was inserted into the right common carotid artery andadvanced into the left ventricle (LV). The catheter was connected to a pressure-conductanceunit (Sigma SA; CD Leycom, Zoetermeer, The Netherlands). The continuous pressure andvolume signals were monitored in real time and digitized at a sample rate of 500/s, usingspecialized software (Conduct NT; CD Leycom, Zoetermeer, The Netherlands) on a notebook-computer. The display of the on-line pressure-volume signals allowed for optimal positioningof the catheter within the left ventricle.

After a short period of stabilization, LV pressure-volume loops were recorded at baseline andthe signals were acquired 3 times for 5 seconds with the ventilator stopped. This yielded a totalof 120150 cardiac cycles from which the following parameters were determined usingspecialized Circlab analysis-software (Leiden University Medical Center, Leiden, TheNetherlands): heart rate (HR), LV end-systolic pressure (Pes), LV end-diastolic pressure (Ped),LV change in positive and negative pressures (dP/d t max and dP/d t min respectively) and LVisovolumic relaxation time constant (Tau). After the steady state measurements, pressure-volume relations were measured 3 times by transiently occluding the inferior vena cava. Theend-systolic pressure-volume relation (ESPVR; or end-systolic elastance: E es) and the slope

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of the dP/d t max with respect to volume (dP/d t max / EDV) were derived from the acquired cardiaccycles.

Histology and immunohistochemistry

After functional analysis, hearts were harvested, perfusion fixed at physiologic pressure withformalin, and embedded in paraffin using standard protocols. 16 Coronal sections were sampledfrom apex to base at 1.0mm intervals and stained with Azan (Sigma) according to themanufactures protocol. Digital photographs were made and infarct size was calculated usingthe following formula: 17 [length of coronal infarct perimeter (epicardial + endocardial)/ totalleft ventricle coronal perimeter (epicardial + endocardial)] 100. Other sections were stainedfor connexin43 (connexin43 rabbit polyclonal antibody, Zymed, South San Francisco, CA)using standard techniques. 16

Calcium transient imaging of peri-infarct myocardium

Hearts were prepared for two photon molecular excitation (TPME) laser scanning microscopyand perfused with oxygenated normal Tyrodes solution containing 50 micromol/Lcytochalasin D as described previously. 18 Images were recorded with a Bio-Rad MRC 1024Laser Scanning microscope modified for TPME. Illumination for 2-photon excitation wasprovided by a mode-locked Ti:Sapphire laser (Spectraphysics, Mountain View, CA); the

excitation wavelength was 810 nm. Hearts were imaged through a Nikon 60 1.2 numericalaperture water-immersion lens with a working distance of 200 microns. Images were collectedat a resolution of 0.43 m/pixel along the xy-plane. For full-frame mode analyses (512 512pixels), hearts were scanned at 1.46 and 0.73 frames per second on horizontal ( x, y ) planes andthe resulting images digitized at 8-bit resolution and stored directly on the hard disk. For line-scan mode analyses, hearts were scanned repetitively along a line spanning at least 2 juxtaposedcardiomyocytes (scan speed was 110 microns/ms at a rate of 32 Hz). Line-scan images werethen constructed by stacking all lines vertically. Post-acquisition analysis was performed usingMetaMorph software version 4.6r (Universal Imaging Incorporation, Downingtown, PA).

Statistical analysis

All data are presented as mean SEM. Between-group comparisons were analysed by unpairedt test. Significance was assumed at P


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Histological analysis and calcium transients of newly formed myocardium

Gross analysis of sections prepared from the non-transgenic hearts revealed that the myocardialcontent of the infracted region remained largely unchanged between 7 days and 180 days postMI (Figure 1a). In contrast, a large portion of the scar tissue present at 7 days post-MI wasreplaced with myocardial tissue by 180 days post-MI in the MHC-cycD2 transgenic mice(Figure 1a). This newly formed myocardium was observed to envelop scar tissue in sectionsprepared from apical regions of the heart (Figure 1a, arrows). Moreover, scar tissue was largelyresolved in sections located near the base of the heart (Figure 1a, arrowheads). Microscopicexamination of the newly formed myocardium at the apical scar/myocardial border of MHC-cycD2 hearts at 180 days post-MI revealed the presence of cardiomyocytes with well-organizedsarcomeres (Figure 1b, left panel). Connexin43 (a major component of the cardiac gap junction)immune reactivity was readily detected between cardiomyocytes in this region (Figure 1b, rightpanel).

The presence of connexin43 immune reactivity at junctional complexes between adjacentcardiomyocytes raised the possibility that the newly formed myocardium might participate ina functional syncytium with the surviving remote myocardium. To directly test this, heartsfrom MHC-cycD2 mice harvested at 180 days post-MI were placed on a Langendorff apparatus, perfused with the calcium sensing dye rhod-2, and imaged using two photonmolecular excitation laser scanning microscopy. This assay permitted direct monitoring of intracellular calcium ([Ca 2+]i) transients in intact hearts. 18 The hearts were imaged at the apicalscar/myocardium border (i.e., the anatomical position of the newly formed myocardium).Periodic increases in rhod-2 fluorescence, due to spontaneous action potential-evokedelevations in cytosolic calcium concentration, were visible as ripple-like wave fronts in thecardiomyocytes, but not in the adjacent scar tissue, in images obtained in TPME full-framemode (Figure 2a).

To better monitor the temporal changes in [Ca 2+]I, fluorescence signals were also recorded inline-scan mode during normal sinus rhythm. The scan line (Figure 2a, white bar) traversedthree cardiomyocytes in the newly formed myocardium. This line was repeatedly imaged andthe resulting line-scans were stacked vertically (Figure 2b). Averaged traces of the intensityof the fluorescence signal from the cardiomyocytes were then generated from the line-scandata (Figure 2c). Cardiomyocytes located in the newly formed myocardium exhibited transientincreases in rhod-2 fluorescence (corresponding to spontaneous action potential-evokedincreases in [Ca 2+]i) in synchrony with one another as well as with the remote myocardium.To examine [Ca 2+]i transient duration, changes in fluorescence for individual cells werenormalized such that 0 represented the fluorescence value prior to [Ca 2+]i transient onset and1 represented the peak fluorescence value. Superimposition of normalized [Ca 2+]i transientsrevealed that [Ca 2+]i transient duration in individual cardiomyocytes within the newly formedmyocardium were similar to one another, and moreover were similar to those recorded inremote cardiomyocytes (Figure 2d). Thus, newly formed myocardium in infarcted MHC-cycD2 transgenic hearts appeared to participate in a functional syncytium with the survivingremote myocardium.

Cardiac function

To determine if the presence of coupled, newly formed myocardium had a positive impact oncardiac function, left ventricular pressure-volume measurements were compared betweensham-operated and infarcted non-transgenic mice, and between sham-operated and infarctedMHC-cycD2 mice, at 7, 60 and 180 days post-surgery. For sham surgery, the chest andpericardium were opened but the coronary artery was not occluded. As expected, MI resultedin a marked and statistically significant deterioration in many physiologic parameters of thenon-transgenic mice at 7 days post-MI as compared to the corresponding sham-operated



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animals (Table 1a). No improvement in cardiac function was apparent at 60 and 180 days post-MI, consistent with the absence of improved cardiac architecture at these time points in thenon-transgenic animals.

Cardiac function was similarly reduced in the MHC-cycD2 hearts at 7 days post-MI ascompared to the sham-operated animals (Table 1b), in agreement with the deterioration of cardiac architecture seen at this time point. However, marked improvements of functional

parameters in the transgenic mice were observed at later time points post-MI (Table 1b).Improvement in the left ventricular peak positive pressure rise rate-end diastolic volume slope(dP/d t max / EDV) was particularly noteworthy, as this parameter provided a highly reproducibleand load-independent index of myocardial contractility. 19 At 180 days post-MI, all parametersmeasured were not statistically different in infarcted MHC-cycD2 mice as compared to MHC-cycD2 mice with sham surgery, indicating a remarkable degree of functional recovery in thetransgenic hearts. The impact of transgene expression on cardiac function was even moreapparent when the parameters measured in infarcted mice were normalized to those in thecorresponding sham-operated animals (Figure 3); all parameters tested in the MHC-cycD2mice improved with time. In contrast, physiologic parameters either did not improve ordeteriorated in the non-transgenic animals.

DiscussionPrevious studies indicated that cardiomyocyte-restricted cyclin D2 expression resulted inregenerative growth in injured hearts, as evidenced by increased cardiomyocyte number andconcomitant reduction of scar tissue mass at 150 days post MI. 12 The data presented heredemonstrated that regenerative cardiac growth was present as early as 60 days post-MI in MHC-cycD2 transgenic mice. Moreover, cardiomyocytes in the newly formed myocardiumexpressed connexin43, and were functionally coupled with one another and with the remotemyocardium as demonstrated by TPME imaging of [Ca 2+]i transients. Cardiac functionalparameters improved in the infarcted MHC-cycD2 hearts, but not in the non-transgenicsiblings, as compared to their respective sham-operated controls. Importantly, the degree of functional improvement in infarcted MHC-cycD2 mice correlated directly with reduction ininfarct size (and concomitantly, the newly formed myocardium content) at 60 vs. 180 dayspost-MI.

To date, only a limited number of proteins have been shown to induce sustained ventricularcardiomyocyte cell cycle activity when expressed in adult transgenic animals. These includeSV40 Large T antigen, 20 an inducible form of c-myc, 21 CDK-2, 22 dominant interferingTSC2, 23 dominant interfering p193, 13 cyclin A2, 24 IGF-1 25 and bcl-2. 26 Of these, onlydominant interfering p193 (an E3 ubiquitin ligase molecule originally identified as an SV40Large T Antigen binding protein) and IGF-1 have so far been shown to improve cardiacfunction following myocardial injury. In the case of the dominant interfering p193 mice,cardiomyocyte cell cycle activity was induced following MI, 13 resulting in more favorablepost-MI ventricular remodeling and a concomitant improvement in cardiac function. 27 In thecase of the IGF-1 mice, it was not clear if the beneficial effect on cardiac function resultedfrom transgene-induced cardiomyocyte proliferation or alternatively from reduced levels of cardiomyocyte apoptosis. 28, 29 Paradoxically, although cardiomyocyte cell cycle activity was

induced in adult mice over-expressing CDK-2, these animals exhibited an aberranthypertrophic response to surgically induced pressure overload. 22 Importantly, none of thesestudies demonstrated progressive restoration of cardiac structure and function post-injury,which would be consistent with regenerative growth of the myocardium.

In contrast, the data presented here indicates that MHC-cycD2 mice undergo a progressiveimprovement in both cardiac structure and function following MI. Given that the MHC-



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promoter is restricted to differentiated cardiomyocytes, and given the high rates of cardiomyocyte DNA synthesis observed previously following MI, the simplest interpretationfor the improvement of cardiac architecture and function observed in the current study istransgene-induced proliferation of pre-existing cardiomyocytes. It is however possible thattransgene expression may also enhance the reparative capacity of putative cardiomyogenicprogenitor cells. For example, adult stem cell-derived cardiomyocytes would also likely exhibitenhanced cell cycle activity, provide that they express alpha myosin heavy chain promoter (and

concomitantly, the MHC-cycD2 transgene). The use of a conditional transgene system isrequired to quantitate the relative contributions of pre-existing cardiomyocytes andcardiomyogenic stem cells observed in the current study.

Regardless of the mechanistic origin of the de novo myocardium observed here, it is noteworthythat the magnitude of cardiac functional improvement following MI in the MHC-cycD2 heartscompared quite favorably to that obtained in most experimental studies using adultcardiomyogenic stem cell-based transplantation or mobilization interventions. Indeed, thereported impact of stem cell intervention on cardiac structure and cardiac function has beenhighly variable, ranging from considerable to no detectable impact, despite the use of similarcell types, injury models, and experimental read-outs. 6, 3037 Importantly, functionalimprovement in the injured MHC-cycD2 transgenic hearts persisted for 180 days post-MI. Itis of interest to note that the degree of infarct reduction in MHC-cycD2 at 180 days post-MI

(35%) was somewhat greater than that observed in our previous study which was analyzed at150 days post-MI).[cite ref 12] This is likely to be attributable at least in part to the longerduration of the experiment.

Modulation of cyclin D activity in cardiomyocytes after myocardial injury may have clinicalimplications for cardiac regeneration. For example, cyclin D2 gene transfer in humanmyocardium could possibly lead to a gene-based regenerative mechanism in patients. Insupport of this, in vivo experiments revealed that genetically nave adult rat cardiomyocytesrespond to cyclin D following adenoviral gene transfer. 38 Although our model utilized a ratherstrong, constitutively active promoter, the level of transgene-derived cyclin D2 expression inthe adult mice were similar to that seen for the endogeneous cyclin D2 gene in fetal heartswhen similar levels of total protein were compared. [cite ref 12 here] Unfortunately, similarlevels of transgene expression were observed in the different MHC-cycD2 lineages that we

generated, precluding the establishment of a dose-response relationship between the level of cyclin D expression and regenerative growth. Although cell cycle activity persisted in agedMHC-cycD2 mice, disorganized tumor-like growth has not been observed and the heartsremained comprised of well-differentiated cardiomyocytes. Nonetheless, use of constitutivelyactive promoters to target growth promoting genes would not be appropriate for therapeuticapplications. Perhaps more realistically, development of pharmacological agents capable of modulating cyclin D expression and/or activity in cardiomyocytes might prove to be a usefulapproach to engender regenerative cardiac growth. Regardless of the mechanism employed,cardiomyocyte cell cycle induction may represent an important therapeutic tool for cardiacregeneration and enhancement of cardiac function after MI.

AcknowledgmentsWe thank Dr. Paul Steendijk (Dept. of Cardiology, Leiden University Medical Center, Leiden, The Netherlands) forassistance with pressure-volume data analysis.

Funding

This work has been supported by the Hein J.J. Wellens Foundation and the Foundation De Drie Lichten, (both in TheNetherlands), and by grants from the Heart Lung and Blood Institute of the National Institutes of Health (USA).



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Figure 1.

Characterization of myocardial infarcts in non-transgenic and MHC-cycD2 mice. (a)Representative Azan staining of coronal sections from non-transgenic and MHC-cycD2 heartsat 7 and 180 days post-MI. The sections were sampled at 2, 3 and 4 mm from the apex of theheart, as indicated. Arrows indicate regions of newly formed myocardium in the MHC-cycD2heart at 180 days post-MI which were not present in the corresponding anatomical location at7 days post-MI, nor in the non-transgenic mice. Arrowheads indicate where the infarct scarhas been largely resolved near the base of the heart by 180 days post-MI in the MHC-cycD2.(b) Consecutive sections of the apical scar/myocardium border of an MHCcycD2 mouse at 180days post-MI stained with Azan (left panel) and connexin-43 (right panel; HRP-conjugatedsecondary antibody). Arrow indicates the junctional complex between two cardiomyocyteswithin the newly formed myocardium; SC, scar; MY, myocardium.



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Figure 2.Characterization of [Ca 2+]i transients in the newly formed myocardium. (a) Full-frame TPMEimage of cells at the apical scar/myocardium border of an MHC-cycD2 heart at 180 days post-MI (i.e., the newly formed myocardium). The white bar demarks the position of line-scan modedata acquisition and numbers indicate the position of individual cardiomyocytes; SC, scar;MY, myocardium. (b) Stacked line-scan image acquired during automatic depolarization fromthe heart depicted in panel (a). (c) Spatially integrated traces of the changes in rhod-2 (red)fluorescence during spontaneous depolarizations from the heart depicted in panel (a). (d)

Superimposed tracings of spontaneous changes in rhod-2 fluorescence as a function of timefrom cardiomyocytes at the scar/myocardium border (upper traces) and from remotely-locatedcardiomyocytes (lower traces). For each cell, spatially averaged changes in rhod-2 fluorescencewere obtained and subsequently normalized such that 0 represents the fluorescence intensitybefore the [Ca 2+]i transient and 1 represents the peak fluorescence intensity. Similar resultswere obtained when the preparations were paced via point stimulation at remote sites of themyocardium.



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Figure 3.Functional parameters in infarcted non-transgenic (open bars) and MHC-cycD2 (black bars)mice, normalized to their respective sham-operated animals. Y-axis: % in sham operationgroups. P


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6 0 d M I

n = 9

1 8 0 d s h a m

n = 9

1 8 0 d M I

n = 8

H R ( b e a

t s / m i n )

5 2 5 7

5 3 4 3 0

5 2 0 1 0

5 1 7 1 0

4 9 4 1 0

4 5 8 1 7

P e s

( m m

H g )

5 7 . 5

1 . 3

5 6 . 7

1 . 8

6 2 . 8

3 . 3

5 9 . 2

2 . 0

5 8 . 6

2 . 8

5 6 . 7

2 . 3

P e d

( m m

H g )

2 . 2 0 . 2

3 . 8 0 . 9

3 . 1 0 . 4

3 . 8 0 . 8

2 . 0 0 . 5

3 . 6 0 . 5 *

d P / d

t m a x

( m m H g / s )

6 1 0 5

. 6 2 2 2

. 9

3 7 3 6

. 6 1 6 9

. 7 *

6 6 2 7

. 1 1 4 7

. 7

3 3 2 2

. 3 1 6 4

. 7 *

5 7 9 4

. 8 3 9 1

. 8

3 3 4 2

. 5 1 0 5

. 9 *

d P / d

t m

i n ( m m H g / s )

3 9 1 6

. 3 8 6 . 8

3 3 2 8 . 8 3 4 5 . 0

4 1 1 7

. 3 1 2 4

2 9 2 7

. 6 2 0 6

. 6 *

3 9 5 0

. 6 2 0 0

. 1

2 8 6 1

. 6 1 3 5

. 7 *

T a u

( m s )

1 0 . 1

0 . 3

1 2 . 5

0 . 7

*

1 0 . 0

0 . 3

1 4 . 2

0 . 8

*

1 0 . 6

0 . 5

1 3 . 8

0 . 5

*

E S P V R ( m m H g / l

)

4 1 . 1

3 . 2

2 3 . 9

2 . 0

*

3 6 . 2

3 . 7

1 4 . 3

1 . 9

*

2 7 . 3

5 . 1

1 6 . 0

3 . 0

d P / d

t m a x

/ E D V

( m m

H g . s -

1 . l -

1 )

2 0 9 5

. 3 2 0 0

. 2

1 0 9 4

. 4 1 5 8

. 8 *

1 7 2 1

. 7 2 0 0

. 0

6 3 6 . 0 1 4 2 . 8 *

1 2 2 9

. 2 1 7 7

. 8

5 2 0 . 2 1 0 6 . 7 *

T a b

l e 1 b

. H e m o d y n a m

i c p a r a m e t e r s

i n M H C - c y c

D 2 t r a n s g e n i c m

i c e a t

7 , 6 0 a n

d 1 8 0 d a y s a f

t e r s h a m - o p e r a

t i o n o r

M I .

7 d s h a m

n = 1 2

7 d M I

n = 1 0

6 0 d s h a m

n = 1 2

6 0 d M I

n = 1 2

1 8 0 d s h a m

n = 1 0

1 8 0 d M I

n = 8

H R ( b e a

t s / m i n )

5 2 4 9

5 0 2 1 1

5 1 6 7

5 2 6 7

4 8 6 1 1

4 9 6 2 0

P e s

( m m

H g )

6 2 . 5

4 . 7

5 8 . 2

3 . 5

6 2 . 2

2 . 1

6 1 . 8

2 . 1

5 9 . 3

2

6 2 . 2

5 . 2

P e d

( m m

H g )

2 . 7 0 . 4

3 . 5 0 . 7

3 . 0 0 . 5

3 . 6 0 . 6

2 . 8 0 . 6

3 . 0 1 . 0

d P / d

t m a x

( m m H g / s )

6 1 3 2

. 5 1 7 4

. 7

3 4 5 9 . 9 2 9 5 . 8 *

6 5 6 3

. 2 1 5 2

. 0

4 1 8 1

. 5 3 3 8

. 9 *

5 8 6 5

. 0 2 1 5

. 5

4 9 6 1

. 8 7 3 1

. 8

d P / d

t m

i n ( m m H g / s )

4 0 7 9

. 8 2 2 8

. 3

3 0 7 7

. 8 3 1 8

. 0 *

4 1 1 7

. 0 7 9 . 5

3 5 2 1

. 3 2 7 0

. 8 *

3 8 9 4

. 8 1 3 1

. 4

3 7 1 9

. 2 3 9 7

. 2

T a u

( m s )

1 0 . 1

0 . 3

1 1 . 9

0 . 3

*

1 0 . 2

0 . 2

1 2 . 2

0 . 4

9 *

1 0 . 9

0 . 4

1 1 . 6

0 . 7

E S P V R ( m m H g / l

)

4 5 . 5

4 . 4

2 6 . 3

5 . 6

*

3 8 . 3

3 . 7

2 9 . 6

2 . 3

3 1 . 2

2 . 8

2 9 . 0

3 . 1

d P / d

t m a x

/ E D V

( m m

H g . s -

1 . l -

1 )

1 8 7 8

. 5 1 1 4

. 1

8 7 3 . 7 1 5 0 . 4 *

1 9 5 2

. 7 1 0 4

. 3

1 3 2 0

. 7 1 1 6

. 7 *

1 3 3 6

. 1 1 1 9

1 1 7 2

. 7 2 2 2

. 7

H R

, h e a r t r a

t e ; P e s , l

e f t v e n

t r i c u l a r e n

d - s y s t o l

i c p r e s s u r e ; P e d , l

e f t v e n t r i c u

l a r e n d - d

i a s t o l

i c p r e s s u r e ; d

P / d t m a x , l

e f t v e n t r i c u

l a r p o s

i t i v e p r e s s u r e r i s e r a

t e ; d

P / d t m

i n , l

e f t v e n

t r i c u l a r p e a k n e g a

t i v e p r e s s u r e

r i s e r a

t e ; T a u , l e f

t v e n

t r i c u l a r

i s o v o l u m

i c r e

l a x a

t i o n

t i m e c o n s

t a n t ; E

S P V R

, e n d - s y s

t o l i c p r e s s u r e - v o l u m e r e

l a t i o n ;

d P / d t m a x

/ E D V

, s l o p e o f

t h e

l e f t v e n t r i c u

l a r p o s i

t i v e p r e s s u r e r i s e r a

t e w

i t h r e s p e c

t

t o v o

l u m e .



13/13





* p

< 0 . 0

5 , M I m i c e v s . t

h e i r r e s p e c

t i v e s h a m - o p e r a

t e d c o n t r o

l s .


Date post:	07-Apr-2018
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Cardiomyocyte Cell

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