Age-related changes in lamin A/C expression in cardiomyocytes

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Age-Related Changes in Lamin A/C Expression in Cardiomyocytes

Running title: Age-Related Changes in Lamin A/C in Cardiomyocytes

Jonathan Afilalo MD 1, Igal A. Sebag MD 2,3, Lorraine E. Chalifour PhD 3,4, Daniel Rivas MSc 4,5, Rahima Akter MD 4, Kamal Sharma MD PhD 2, Gustavo Duque MD PhD 4,5,6

1 Division of Internal Medicine, Department of Medicine, Sir Mortimer B. Davis Jewish General Hospital, Montréal, McGill University, Québec, Canada; 2 Echocardiography Laboratory, Division of Cardiology, Department of Medicine, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montréal, Québec, Canada;

3 Bank of Montreal Center for the Study of Heart Disease in Women, Lady Davis Institute for Medical Research, Montréal, Québec, Canada; 4 Division of Experimental Medicine, Department of Medicine, McGill University, Montréal, Québec, Canada; 5 Bloomfield Center for Studies in Aging, Lady Davis Institute for Medical Research, Montréal, Québec, Canada; 6 Division of Geriatric Medicine; Department of Medicine, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montréal, Québec, Canada.

Address for correspondence:Gustavo Duque, MD, PhD

Division of Geriatric Medicine, SMBD-Jewish General Hospital Bloomfield Centre for Studies in Aging, Lady Davis Institute for Medical Research

3755 Côte Sainte Catherine Montréal, Québec, Canada H3T 1E2

Phone: (514) 340-7501 Fax: (514) 340-7547

Email: [email protected]

This project was presented at the American Geriatrics Society Annual Meeting (Chicago, IL, 2006).

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Copyright Information

Articles in PresS. Am J Physiol Heart Circ Physiol (May 25, 2007). doi:10.1152/ajpheart.01194.2006

Copyright © 2007 by the American Physiological Society.

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ABSTRACT

Lamin A and C (A/C) are type V intermediate filaments that form the nuclear lamina.

Lamin A/C mutations lead to reduced expression of lamin A/C and diverse phenotypes such

as familial cardiomyopathies and accelerated aging syndromes. Normal aging is associated

with reduced expression of lamin A/C in osteoblasts and dermal fibroblasts but has never

been assessed in cardiomyocytes. Our objective was to compare the expression of lamin A/C

in cardiomyocytes of old (24 months) versus young (4 months) C57Bl/6J mice using a well

validated mouse model of aging. Lamin B1 was used as a control. Immunohistochemical and

immunofluorescence analyses showed reduced expression of lamin A/C in cardiomyocyte

nuclei of old mice (proportion of nuclei expressing lamin A/C, 9% vs. 62%, p<0.001). Lamin

A/C distribution was scattered peripherally and perinuclear in old mice, whereas it was

homogeneous throughout the nuclei in young mice. Western blot analyses confirmed reduced

expression of lamin A/C in nuclear extracts of old mice (ratio of lamin A/C:B1, 0.6 vs. 1.2,

p<0.01). Echocardiographic studies showed increased left ventricular wall thickness with

preserved cavity size (concentric remodeling), increased left ventricular mass, and a slight

reduction in fractional shortening in old mice. This is the first study to show that normal

aging is associated with reduced expression and altered distribution of lamin A/C in nuclei of

cardiomyocytes.

KEYWORDS

aging, lamin, laminopathy, nucleus, cardiomyocyte, cardiomyopathy

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INTRODUCTION

Lamin A and C (A/C) are type V intermediate filaments encoded by the LMNA gene

that form the nuclear lamina (17). The functions of lamin A/C are to support the inner

nuclear envelope and to participate in DNA repair, signal transduction, mesenchymal stem

cell differentiation, mitosis, and apoptosis (9; 17; 24). LMNA mutations (“laminopathies”)

lead to a reduction in lamin A/C expression and diverse phenotypes such as familial

cardiomyopathy and the Hutchison-Gilford Progeria accelerated aging syndrome (5; 10; 33).

Normal aging is associated with a reduction in lamin A/C expression in mouse osteoblasts

and human dermal fibroblasts (9; 30). To date, the effect of normal aging on lamin A/C

expression in cardiomyocytes remains unknown.

Age-related changes in lamin A/C expression in cardiomyocytes may be associated

with the downstream changes in myocardial function and structure seen in aging hearts. This

is suggested by observations of marked myocardial abnormalities in LMNA mutation carriers

and in lamin A/C deficient mice (26; 35). We hypothesized that normal aging is associated

with a reduction in lamin A/C expression in cardiomyocytes. We used lamin B1 expression

(a non-developmentally regulated protein from the same family found in all nucleated cells

independent of aging) as a control. Our primary objective was to compare the expression of

lamin A/C in cardiomyocyte nuclei of old versus young mice using a well validated mouse

model of aging (13; 19; 20).

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MATERIALS AND METHODS

Animal tissue preparation

C57BL/6J mice (Jackson Laboratory, Bar Harbour, ME, USA) were housed in a

limited access room restricted to aging mice (light:dark 12h:12h) as previously described (9).

All animal manipulations adhered to the Canadian Council on Animal Care Standards and all

protocols were approved by the McGill University and Lady Davis Institute Animal Care

Utilization Committee. The colony was free of any parasitic, bacterial, or viral pathogens as

determined by a sentinel program. Using CO2, animals were euthanized at 4 months of age

(n=5) and at 24 months of age (n=5). Hearts were dissected under sterile conditions, rinsed in

0.01 M PBS, blotted dry, and weighed. Heart pieces were frozen at -80oC for further protein

expression analyses. Tissue was also fixed in 10% formaldehyde for histological analysis.

Several sections of heart (4–5 µm thick) were prepared and stained with hematoxylin and

eosin (H/E) and visualized by light microscope.

Echocardiography

2D/M-mode echocardiography was performed in young (4 months) and old (20

months) C57Bl/6J male mice anesthetised with isoflurane. Mice were placed in an induction

chamber, anesthetized in 2% isoflurane mixed with air then maintained in 0.5-0.7%

isoflurane. After removal of the thoracic fur, mice were placed in a left cubital position.

Echocardiographic parasternal short-axis views at the midventricular level were acquired

using a i13L transducer and a digital ultrasound system (Vivid 7, GE Medical Systems) at a 1

cm depth and 100 frames/sec. Measurements were performed off-line with the use of a

customized version of the EchoPac Software (GE Medical Systems). End-diastolic and end-

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systolic dimensions of the left ventricular (LV) cavity as well as anterior and posterior

thickness of the LV wall were measured in M-mode tracings according to the leading edge

method (27). Measurements were averaged from three consecutive beats of three image

acquisitions. Fractional shortening, relative wall thickness, and LV mass were derived as

described previously (32; 34).

Quantification of lamin A/C and B1 expression by immunohistochemistry

After dissection and fixation, young and old hearts samples were embedded in low-

melting-point paraffin in a Shandon Citadel 2000 automatic tissue processor (Shandon

Scientific Limited, Runcorn, UK). Coronal and transverse sections (4 mm) were mounted on

silane-coated glass slides (Fischer Scientific, Springfield, NJ, USA). Paraffin was removed

with three washes of xylene and rehydrated with washes of graded ethanol (80%–50%–30%)

and PBS. Non-specific binding was blocked by addition of goat serum for 1 hour. Sections

were then incubated with mouse monoclonal IgM lamin A/C antibody (Santa Cruz

Biotechnology, Santa Cruz, CA, USA) for either 4 hours at room temperature or 8–24 hours

at 4°C. Sections incubated with mouse monoclonal IgG lamin B1 antibody (Santa Cruz

Biotechnology, Santa Cruz, CA, USA) were used as controls. After washing with PBS,

hydrogen peroxide complexed rabbit anti-mouse IgG were added to the sections at room

temperature for 30 minutes, followed by a 30 minute incubation with 0.6% hydrogen

peroxide ± chromogen. Immunohistochemical staining was performed using the human ABC

staining system (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Lamin positive cells

showed a brown nucleus with punctate brown staining from the peroxidase-labelled antibody

and blue counterstaining from the hematoxylin. Lamin A/C expression was calculated as the

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number of cardiomyocyte nuclei positively stained for lamin A/C (brown stained nuclei)

divided by the total number of cardiomyocyte nuclei (brown and blue stained nuclei) in each

of 10 randomly chosen high power fields (hpf).

Quantification of lamin A/C by immnunofluorescence

Heart sections were treated as described above, omitting the final step involving

treatment of cells with hydrogen peroxide. After fixation in 4% paraformaldehyde, sections

were washed with PBS and then incubated in PBS with 10% blocking serum for 20 minutes

to suppress non-specific binding of IgG. Sections were incubated with mouse monoclonal

IgM lamin A/C antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with 1.5%

blocking serum overnight at 4oC and then incubated with fluorescein-conjugated secondary

antibody (FITC-Santa Cruz, Santa Cruz, CA, USA) diluted to 2 µg/ml in PBS with 1.5%

blocking serum for 45 minutes. Nuclei were counterstained using propridium iodine (2

µg/ml). Control slides were incubated with rabbit IgG according to manufacturer

instructions, and triplicate tests and control slides were included in immunodetection. Lamin

A/C positive cells showed intense nuclear green fluorescence while negative controls showed

only faint nuclear or cytoplasmic fluorescence. The number of positive cardiomyocyte nuclei

divided by the total number of cardiomyocyte nuclei in each field was calculated as described

above.

Quantification of lamin A/C and B1 expression by western blot

Nuclear extracts were obtained after suspending the heart pieces in 2 volumes of

buffer containing 10 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and protease

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inhibitor cocktail diluted according to the manufacturers instruction (Complete™ protease

inhibitor, Boehringer Mannheim, Laval, QC, Canada). The homogenate was clarified by

centrifugation at 25,000 × g for 20 minutes at 4°C and the nuclear pellet was resuspended in

20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5

mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol. Following a further 20 minute

centrifugation at 25,000 × g, nuclear extracts (supernatant) were dialyzed for 5 hours against

20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM

phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol. Protein content was determined

with a protein assay kit (Bio-Rad, Mississauga, ON, Canada) and samples were then

aliquoted and stored at -80°C. For western blot analyses, nuclear extracts were resuspended

in SDS electrophoresis buffer (Bio-Rad, Hercules, CA, USA), proteins were separated on

SDS-polyacrylamide gels and the proteins electrotransfered to Immobilon P polyvinylidene

difluoride membranes. After blocking with PBS containing 0.1% Tween 20 and 10% non-fat

dry milk, membranes were incubated overnight at 4°C using a monoclonal antibody directed

against lamin A/C and a second monoclonal antibody against lamin B1 (Santa Cruz, Santa

Cruz, CA, USA). Specific staining was revealed after washing and incubating with

horseradish peroxidase-conjugated IgG rabbit anti-mouse antibodies followed by enhanced

chemiluminescence using Lumi-GLO reagents (Kirkegoard & Perry, Gaithensburg, MA,

USA). The lamin A/C signals were quantified by densitometry and normalized according to

lamin B1 signals.

Statistical analysis

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All results are expressed as mean ± standard error of the mean of three replicate

determinations, and statistical comparisons are based on one-way analysis of variance

(ANOVA) or student t-tests. A p-value of <0.05 was considered significant and ≥0.05 was

considered non-significant (NS).

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RESULTS

The young (n=5) and old (n=5) C57BL/6J mice were representative of the litter and

did not have identifiable diseases. Dissection of the murine hearts revealed that the mean

mass of the isolated hearts was 189 ± 15 mg in old mice and 167 ± 15 mg in young mice

(p<0.05). Microscopic inspection of the H&E stained sections did not reveal any gross

pathology in the cardiac tissues (Figure 1 A and B). The number of cardiomyocytes per hpf

was inferior in old mice compared to young mice (40-62 cells/hpf vs. 65-80 cells/hpf,

p<0.05) whereas the cell size was similar (108 µm vs. 110 µm, p=NS).

Echocardiographic results are shown in Table 1. In comparison to young mice, old

mice demonstrated increased LV wall thickness with preserved LV cavity size resulting in

increased relative wall thickness (0.38 ± 0.05 vs. 0.29 ± 0.06, p<0.05) suggestive of

concentric remodeling. Accordingly, LV mass was increased in old mice (121 ± 19 mg vs. 93

± 16 mg, p<0.05). Fractional shortening was slightly reduced in old mice (46.3 ± 3.9% vs.

48.3 ± 3.8%, p<0.05).

Immunohistochemical analyses showed that the expression of lamin A/C but not

lamin B1 was reduced in cardiomyocyte nuclei of old mice (Figure 1 C, D, E, and F). The

proportion of nuclei positively stained for lamin A/C was 9 ± 6% in old mice and 62 ± 16%

in young mice (p<0.001). In contrast, the proportion of nuclei positively stained for lamin B1

control remained constant (90 ± 6% in old mice and 96 ± 4% in young mice, p=NS).

Immunofluorescence analyses also showed that the expression of lamin A/C was reduced in

cardiomyocyte nuclei of old mice (Figure 2 A-D).

In addition to quantitative changes in lamin A/C expression, qualitative changes in

lamin A/C distribution were observed. The distribution of lamin A/C was homogeneous

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throughout the nuclei in young mice, whereas it was scattered towards the periphery and

perinuclear with minimal contact between staining sites in old mice (Figure 2 E and F).

Western blot analyses confirmed and quantified the reduction in lamin A/C

expression in cardiomyocyte nuclear extracts of old mice (Figure 3). The ratio of lamin

A/C:B1 as measured by densitometry was 0.6 in old mice and 1.2 in young mice (p<0.01).

Finally, changes in lamin A/C expression were observed in other heart cells.

Specifically, immunohistochemical analyses suggested that the expression of lamin A/C but

not B1 was reduced in vascular endothelial cell nuclei of old mice compared to young mice

(Figure 4).

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DISCUSSION

Our study is the first to show that normal aging is associated with a reduced

expression of lamin A/C in cardiomyocytes. Moreover, we found that normal aging is

associated with a scattered perinuclear distribution of lamin A/C and may be associated with

a reduced expression of lamin A/C in vascular endothelial cells. Thus, age-related changes in

lamin A/C expression and distribution denote a novel aging mechanism previously described

in the dermatologic and osteoarticular systems (9; 30) and now discovered in the

cardiovascular system.

Our finding of reduced myocardial lamin A/C expression with aging is preceded by

the well documented finding of reduced myocardial lamin A/C expression with inherited

LMNA mutations (1; 39). LMNA mutations are among the most common causes of familial

autosomal-dominant cardiomyopathy (18). Individuals with these mutations often have heart

failure with increased LV wall thickness (37). Up to 88% of affected individuals have

electrophysiological disturbances such as sick sinus syndrome, atrioventricular block, and

atrial fibrillation or flutter (10; 16). This constellation of findings shares several features with

the physio-pathological changes seen in the aging heart (22; 23). We speculate that LMNA-

related familial cardiomyopathy and age-related senile cardiomyopathy may represent two

entities in a spectrum of lamin A/C deficient heart disease.

The newly discovered association of aging and myocardial lamin A/C expression is a

fundamental first step in lamin-cardiology research. It opens the door for further

characterization of the age-related changes in myocardial lamin A/C expression and

distribution. More importantly, it opens the door for mechanistic research to test whether

these exists a causal link between the observed decline in lamin A/C and the parallel

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abnormalities in myocardial structure and function. One potential link between lamin A/C

and aging hearts may be that both are epitomized by an impaired cellular and nuclear

response to stressors. The structural model of age-related changes in lamin A/C suggests that

loss of lamin function causes nuclear fragility which leads to permanent damage or death in

the face of mechanical or environmental stressors (33). Similarly, age-related changes in the

heart are described as a state of fragility or reduced adaptation to acute and chronic stressors

such as exercise or myocardial ischemia (11; 13-15; 21; 27; 29).

In agreement with prior studies conducted in animal models and in human subjects

(2; 3; 6; 31; 40), old mice demonstrated increased LV wall thickness with preserved cavity

size resulting in increased relative wall thickness (concentric remodeling) and mass. Systolic

function was slightly, yet significantly, reduced as previously demonstrated by Yang et al

(40). Although diastolic function was difficult to assess given the rapid heart rates of our

mice under physiologic conditions (mean 548-563) and the challenge of transducer

positioning for reliable parallel mitral inflow, our finding of concentric hypertrophy is

consistent with the finding of a relaxation abnormality in senescent mice demonstrated by

Taffet et al (36). We do not intend for the echocardiographic data to be causally explanatory;

however, we believe that these data add an important dimension to our study by showing that

the morphological and functional correlation of our histopathological findings are consistent

with the expected changes of myocardial aging.

Clinically, lamin A/C has the potential to be a prognostic marker and a therapeutic

target. Among 15 patients with nonischemic cardiomyopathy requiring left ventricular assist

device support, lamin A/C expression over time was a strong predictor of myocardial

recovery leading to explantation of the device (4). In presymptomatic LMNA mutation

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carriers, lamin A/C expression may be used as a screening tool to identify high-risk subjects

who may benefit from more aggressive therapy (28). Therapeutic agents such as

farnesylation modulators have been shown to prevent or reverse some of the nuclear defects

in Hutchison-Gilford Progeria Syndrome (7; 8; 12; 25; 38). These agents modulate post-

translational conversion of precursor prelamin A to the active lamin A correcting the nuclear

defect associated with lamin A depletion or prelamin A accumulation. To our knowledge, the

effect of farnesylation modulators on normal aging or on the cardiovascular system has not

been evaluated.

In conclusion, the expression of lamin A/C is substantially reduced and the

distribution is scattered peripherally in nuclei of cardiomyocytes isolated from a validated

mouse model of aging. Further research in this field may attempt to clarify the causal link

between lamin A/C and aging hearts, and to explore the value of farnesylation modulators as

novel therapeutic agents to counter the potentially negative effects of lamin A/C depletion on

the heart.

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ACKNOWLEDGEMENTS

We would like to thank Dr. Ernesto Schiffrin (Chief, Department of Medicine,

SMBD-Jewish General Hospital) for his diligent review, and Dr. Alexander Marcus

(Department of Pathology, St. Mary’s Hospital) for his assistance in examining the

histological sections. This work was supported by operating grants from the Canadian

Institutes for Health Research and the Heart and Stoke Foundation of Quebec. Dr. Duque

holds a bursary from the Fonds de la Recherche en Santé du Québec.

DISCLOSURES

The authors have not published or submitted any related papers from the same study

and have no conflicts of interest or financial disclosures to report.

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FIGURE LEGENDS

Figure 1:

Microscopic inspection of the H&E stained sections in young (A) and old (B) mice did not

reveal any gross pathology in the cardiac tissues. The number of cardiomyocytes per hpf was

greater in young mice compared to old mice whereas the cell size was similar.

(C) Lamin A/C expression in young cardiomyocytes, (D) Lamin B1 expression in young

cardiomyocytes, (E) Lamin A/C expression in old cardiomyocytes, (F) Lamin B1 expression

in old cardiomyocytes. Note that the expression of lamin A/C but not B1 is decreased in old

cardiomyocytes. The scatter plot (G) shows the proportion of nuclei positive for lamin A/C

in each of the 10 high power fields analyzed per mouse (9% vs. 62%, p<0.001).

Figure 2:

Imunofluorescence staining of lamin A/C in young cardiomyocytes (A) vs old

cardyomyocytes (C). Young cardiomyocytes show bright fluorescence staining at the nuclei

(A, white arrows) by lamin A/C antibody. Lamin A/C labeling is reduced in old

cardiomyocytes (B, white arrows). Panels A and C show overlap between propidium iodine

and green immunofluorescence. Panel B and D show PI counterstaining to determine the

total number of nuclei in the field.

(E) Lamin A/C distribution in young cardiomyocytes, (F) Lamin A/C distribution in old

cardiomyocytes. Note that the distribution of lamin A/C as shown by the red arrows is

peripherally scattered and perinuclear in old vs. homogeneous and intranuclear in young

cardiomyocytes.

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Figure 3: Quantification of Lamin A/C and Lamin B1 (Control) by Western Blot

(Left) Western blot of lamin A/C and B1 expression in nuclear extracts of young

cardiomyocytes, (Right) Western blot of lamin A/C and B1 expression in nuclear extracts of

old cardiomyocytes. One representative blot of each protein (60 µg) is shown from three

experiments that yielded similar results. The signals were quantified by densitometry and

normalized according to the lamin B1 expression. The bar graph shows the ratio between

lamin A/C : lamin B1 which is significantly decreased in old cardiomyocytes (0.6 vs. 1.2,

p<0.01).

Figure 4: Vascular Expression of Lamin A/C

(A) Lamin A/C expression in the vascular endothelial cells of young cardiomyocytes, (B)

Lamin A/C expression in the vascular endothelial cells. Note that the expression of lamin

A/C as shown by black arrows is decreased in the vascular endothelial cells of old mice.

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Table 1. Echocardiographic data for young and old mice

Young Old HR (beats/min) 563±72 548±57 LVEDD (mm) 42.5±3.7 41.2±2.8 LVESD (mm) 22.0±2.6 22.0±1.9 SW (mm) 5.7±1 7.3±1.1* PW (mm) 6.6±1.1 8.3±1.3* RWT 0.29±0.06 0.38±0.05* FS (%) 48.3±3.9 46.3±3.8* LVM (mg) 93±16 121±19*

Significance is indicated by an * where p < 0.05 when young versus old values are compared.

Abbreviations: HR, heart rate; LVEDD, left ventricular end diastole dimension; LVESD, left

ventricular end systole dimension; SW, septal wall thickness; PW, posterior wall thickness;

RWT, relative wall thickness calculated as (PW+SW)/LVEDD; FS, fractional shortening

calculated as [(LVEDD-LVESD)/LVEDD] x 100; LVM, left ventricular mass calculated as

1.055 [(PW+SW+LVEDD)3-(LVEDD)3].

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Figure 1: Microscopic inspection of the H&E stained sections in young (A) and old (B) mice did not reveal any gross pathology in the cardiac tissues. The number of

cardiomyocytes per hpf was greater in young mice compared to old mice whereas the cell size was similar. (C) Lamin A/C expression in young cardiomyocytes, (D) Lamin B1

expression in young cardiomyocytes, (E) Lamin A/C expression in old cardiomyocytes, (F) Lamin B1 expression in old cardiomyocytes. Note that the expression of lamin A/C but not B1 is decreased in old cardiomyocytes. The scatter plot (G) shows the proportion of nuclei

positive for lamin A/C in each of the 10 high power fields analyzed per mouse (9% vs. 62%, p<0.001).

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Figure 2: Imunofluorescence staining of lamin A/C in young cardiomyocytes (A) vs old cardyomyocytes (C). Young cardiomyocytes show bright fluorescence staining at the nuclei (A, white arrows) by lamin A/C antibody. Lamin A/C labeling is reduced in old cardiomyocytes (B, white arrows). Panels A and C show overlap between propidium

iodine and green immunofluorescence. Panel B and D show PI counterstaining to determine the total number of nuclei in the field. (E) Lamin A/C distribution in young

cardiomyocytes, (F) Lamin A/C distribution in old cardiomyocytes. Note that the distribution of lamin A/C as shown by the red arrows is peripherally scattered and

perinuclear in old vs. homogeneous and intranuclear in young cardiomyocytes.

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Figure 3: Quantification of Lamin A/C and Lamin B1 (Control) by Western Blot (Left) Western blot of lamin A/C and B1 expression in nuclear extracts of young

cardiomyocytes, (Right) Western blot of lamin A/C and B1 expression in nuclear extracts of old cardiomyocytes. One representative blot of each protein (60 µg) is shown from

three experiments that yielded similar results. The signals were quantified by densitometry and normalized according to the lamin B1 expression. The bar graph shows

the ratio between lamin A/C : lamin B1 which is significantly decreased in old cardiomyocytes (0.6 vs. 1.2, p<0.01).

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Figure 4: Vascular Expression of Lamin A/C (A) Lamin A/C expression in the vascular endothelial cells of young cardiomyocytes, (B) Lamin A/C expression in the vascular endothelial cells. Note that the expression of lamin A/C as shown by black arrows is

decreased in the vascular endothelial cells of old mice.

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Age-related changes in lamin A/C expression in cardiomyocytes

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