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Cardiovascular Research 2000 45(4):961-970; doi:10.1016/S0008-6363(99)00409-5
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Copyright © 2000, European Society of Cardiology

Immunolocalization of annexins IV, V and VI in the failing and non-failing human heart*

Rosalia G Matteo1 and Christine Schomisch Moravec*

Center for Anesthesiology Research, The Cleveland Clinic Foundation and Department of Biology, Cleveland State University, 9500 Euclid Ave, FF40, Cleveland, OH 44195, USA

* Corresponding author. Tel.: +1-216-445-5045; fax: +1-216-445-4658 moravec{at}ccf.org

Received 6 August 1999; accepted 2 November 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
The failing human heart is characterized by changes in the expression and function of proteins involved in intracellular Ca2+ cycling, resulting in altered Ca2+ transients and impaired contractile properties of cardiac muscle. The role of the cardiac annexins in this process remains unclear. Annexins may play a role in the regulation of Ca2+ pumps and exchangers on the sarcolemma, and have been shown to be altered in some cardiac disease states. Objective: The goal of this study was to compare the immunolocalization and expression of annexins IV, V and VI in failing and non-failing human hearts. Methods: We used immunostaining to identify the subcellular location of annexins IV, V and VI proteins within the myocardial cell, and Western blot analysis to quantify the proteins in the same hearts. Results: Annexin IV showed a cytoplasmic distribution in both failing and non-failing human heart cells. Annexin V was localized at the z-line, around lipofuscin granules, and in the cytosol in the non-failing heart cells. Annexin VI was localized at the sarcolemma and intercalated disc. Protein levels of annexins IV and V were up-regulated in failing human hearts, while the expression of annexin VI was unchanged. Conclusions: Alterations in the intracellular localization of annexins, along with up-regulation of annexins IV and V in the failing human heart cells, suggests differential regulation of these Ca2+ regulatory proteins during heart failure.

KEYWORDS Ca-pump; Calcium (cellular); Cardiomyopathy; Heart failure; Sarcolemma


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
In an attempt to elucidate the intracellular mechanisms associated with contractile dysfunction in congestive heart failure, a number of laboratories have investigated the processes of excitation–contraction coupling in failing and non-failing human hearts. Studies have demonstrated altered levels of intracellular Ca2+ [1] impaired sarcoplasmic reticulum function [2,3], altered transcripts for Ca2+ regulatory proteins [4–6], a more controversial change in the corresponding proteins [7], altered protein phosphorylation [8,9] and changes in ion channels [10,11] in the failing human heart.

In spite of the number of studies which have been devoted to Ca2+ cycling in heart failure, only recently has attention focused on the annexins, a novel family of Ca2+-binding proteins which are abundant in the heart and may play an important role in cardiac excitation–contraction coupling. Ca2+-binding proteins, such as calmodulin, troponin C and the annexins, are intermediates for the Ca2+ signal, regulating the function of other proteins involved in excitation–contraction coupling.

Annexins are a family of structurally related proteins with 13 members (annexins I–XIII) identified to date [12,13]. Within this family, annexins IV, V and VI have been implicated in the regulation of channels, exchangers, and intracellular signaling processes in various cell types [14–19]. Annexin IV has been found to inhibit the Ca2+ activated chloride conductance in epithelial cells [14] and is itself a substrate for phosphorylation by protein kinase C [14]. Annexin V has been demonstrated to form voltage-gated cation-selective channels in phospholipid bilayers [15], to mediate Ca2+ movement into phospholipid vesicles [16], to be involved in ANF secretion [20] and to be a high affinity inhibitor of protein kinase C [21,22]. Annexin VI has been shown to regulate the skeletal muscle SR Ca2+ release channel [17,23], to increase conductance through the L-type Ca2+ channel [24] and to modulate activity of the cardiac Na+/Ca2+ exchanger [18]. In the heart, studies have suggested the presence of these three annexins, as well as annexin II and possibly annexins III and VIII [25–29].

The importance of annexins in the cardiovascular system has not been established. All members of the family are anti-coagulants [12]. Preliminary studies have suggested a role for the annexins in ischemia–reperfusion injury, where they may regulate phospholipid homeostasis [27] or mediate increased Ca2+ influx [30]. Annexins III, IV and V have been shown to increase in the plasma of patients following myocardial infarction, presumably being released from injured heart cells [31] or possibly from vascular endothelial cells [12]. Gunteski-Hamblin and colleagues have recently shown that overexpression of annexin VI in the heart results in decreased intracellular Ca2+ cycling and impaired contractility [19], suggesting a role for at least one of the cardiac annexins in excitation–contraction coupling.

In pathophysiological states, the relative amount of annexins found in the heart may be altered. Work by Song and co-workers, examining annexin levels in the explanted hearts of cardiac transplant recipients, has demonstrated a significant increase in the expression of annexin V and a decrease in the expression of annexin VI [29]. These data are provocative and suggest that the annexins, like other Ca2+ regulatory proteins, may be altered in human heart failure.

In the current study, we have sought to extend the observations of Song and colleagues to the intracellular localization of the annexins. We have examined the protein levels of annexins IV, V and VI in failing and non-failing human hearts, and have used immunolocalization to compare their subcellular distribution. Results presented here confirm their findings that the annexins are differentially regulated in human heart failure, and further demonstrate that this regulation involves not only the steady-state amounts of the proteins, but also their localization.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Tissue source
Twelve explanted human hearts were obtained from patients undergoing cardiac transplantation at the Cleveland Clinic Foundation. Six non-failing human hearts were obtained from unmatched organ donors, as previously described [32]. Of the patients with heart failure, five had a diagnosis of end stage ischemic heart disease (ICM), and seven had a diagnosis of idiopathic dilated cardiomyopathy (DCM). Characteristics of individual patients are summarized in Tables 1 and 2Go. All hearts were arrested with cold cardioplegic solution in the operating room (composition in mmol/l: NaCl 147.2, MgCl2 16.0, KCl 20.0, NaHCO3 10.0 and CaCl2 2.25) and were immersed in the same solution for transport to the laboratory. Time between explant of the heart and arrival in the laboratory did not exceed 30 min for failing hearts and 90 min for non-failing hearts (due to time in transit from the donor institution). Once in the laboratory, tissue was immediately separated by heart chamber, frozen in liquid nitrogen, and stored at –80°C until use.


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  		Table 1 Characteristics of the patients from whom failing hearts were obtaineda

 

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  		Table 2 Characteristics of the patients from whom non-failing hearts were obtaineda

 
The lung, liver, kidney and skeletal muscle samples were specimens collected by the Department of Pathology at the Cleveland Clinic Foundation at the time of routine surgical procedures. All protocols described in this manuscript were approved by the Institutional Review Board of the Cleveland Clinic Foundation and the investigation conforms with the principles outlined in the Declaration of Helsinki.

2.2 Immunocytochemistry
Endocardial trabecular muscles were dissected from the left ventricle and right atrium of each heart. Muscles were secured on small pieces of wax and frozen in liquid nitrogen. The frozen muscles were stored at –80°C until preparation for sectioning.

Frozen longitudinal sections (4 µm) were cut from the surface of each muscle, using a cryostat (Shandon A5620, Analytical instruments, Plymouth, MN). These sections were used for all immunocytochemical reactions.

After fixation in acetone at –20°C for 20 min, the sections were air dried, rinsed in phosphate buffered saline (PBS), incubated at room temperature with blocking buffer (PBS, containing 2% goat serum, 5% Triton X-100, 5% glycine) for 1 h and then incubated with the primary antibody, 1:25 mouse anti-annexin IV clone 4 (Transduction Laboratories, Lexington, KY), 1:25 goat anti-annexin V R-20 (Santa Cruz Biotechnology, Santa Cruz, CA), and 1:150 mouse anti-annexin VI clone 73 (Transduction Laboratories, Lexington, KY), diluted in PBS containing 2% goat serum, 0.5% Triton X-100 and 5% glycine, at room temperature for 1 h (annexin VI) or overnight at 4°C (annexin IV and V). After rinsing with PBS, the secondary antibody, diluted 1:250 in the same buffer, was applied and incubated at room temperature for 1 h in the dark. The detection system was fluorescein isothiocynate (FITC) labelled antimouse IgG antibody (Vector Laboratories, Burlingame, CA) or anti-goat IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). The sections were rinsed again with PBS and mounted with Vectashield® mounting medium containing propidium iodide to label the nuclei (Vector Laboratories). Slides were examined using a Leica Confocal Laser Scanning microscope.

2.3 Western blot analysis
Human lung, liver, kidney, skeletal muscle and heart left ventricle and right atrium, devoid of fibrotic or adipose tissue, were homogenized in a threefold volume of glycerol buffer at pH 7.4 for 3x30 s using a Polytron (model PT 10/35, Brinkman Instruments, Westbury, NY) homogenizer at 4°C. Protein concentration was determined according to the method of Bradford [33] with bovine serum albumin as the standard. Homogenates were stored at –80°C until use.

Samples were solubilized in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) for 5 min at 95°C. Equal amounts of protein from each sample were subjected to SDS–PAGE using the Laemmli buffer system [34] in a Mini-Protean II Dual Slab Cell (Bio-Rad Laboratories). All samples were run until complete elution of the dye front.

Proteins were transferred to nitrocellulose in a Mini Trans-Blot Transfer Cell (Bio-Rad Laboratories), according to the procedure of Towbin [35]. The transfer was completed at 4°C at a constant voltage setting of 100 V for 1 h. The blots were blocked in 5% gelatin diluted in Tris-buffered saline (TBS), composition: 20 mmol/l Tris–HCl, pH 7.4, 150 mmol/l NaCl, for 1 h at room temperature. The blots were washed three times for 5 min each and were incubated in the primary antibody solution diluted 1:500 in TBS, containing 0.1% gelatin, overnight at 4°C. The same primary antibodies were used for both Western blots and immunolocalization (described above). The blots were washed three times for 15 min in TBS and were incubated in secondary antibody at a dilution of 1:10 000 in the same buffer as above for 1 h at room temperature. The blots were again washed three times for 15 min, and developed using the Vistra ECF Western blotting system (Amersham, Chicago, IL). Proteins were quantified using the Molecular Dynamics Storm Imager and ImageQuant PC software (Molecular Dynamics, Sunnyvale, CA). For each band, normalization was performed by the value of a non-failing heart used as a standard on all blots. The secondary antibodies used for Western blots were: anti-mouse IgG alkaline phosphatase conjugated (Vistra systems) and rabbit F(ab')2 anti-goat IgG alkaline phosphatase conjugated (Southern Biotechnology Associates, Birmingham, AL).

2.4 Statistical analysis
Protein quantitation data are expressed as mean±S.E.M. Comparison between non-failing and failing human tissue was performed by unpaired t-test (NF vs. ICM and NF vs. DCM) or Mann–Whitney test. A value of P<0.05 was accepted as evidence that the groups were significantly different from each other.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
As a positive control for the specificity of the primary antibodies used in this study, we performed Western blot analysis using a variety of human tissues samples. Fig. 1 shows Western blots of left ventricular tissue from the heart (HR), skeletal muscle (SK), liver (LI), kidney (KD) and lung (LN). Anti-annexin IV antibody recognized a major band at approximately 34 kD identified as annexin IV, and a minor band of approximately 68 kD, which may represent a cross reaction with the annexin VI protein. Anti-annexin V antibody recognized a specific band of approximately 32.5 kD in all tissues. The other bands observed in the blot are due to non-specific binding of the secondary antibody and were also present when the blot was incubated with secondary antibody alone (data not shown). Anti-annexin VI antibody specifically recognized a band at approximately 68 kD in all tissues studied.


Figure 1
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  		Fig. 1 Western blot analysis of total homogenate from human heart (HR; left ventricle), skeletal muscle (SK), liver (LI), kidney (KD) and lung (LN). A 30 µg amount of protein from each homogenate was subjected to SDS–PAGE followed by Western blotting. The blots were incubated with anti-annexin IV (panel A), anti-annexin V (panel B) or anti-annexin VI (panel C) and then with the corresponding secondary antibodies, and were visualized using the Vistra system. Quantification was performed using a Storm imager.

 
3.1 Annexin IV
For the immunolocalization studies, one representative failing heart is compared to one representative non-failing heart. Among the failing hearts, there were no differences in the location of any of the annexins between DCM and ICM hearts. When the heart sections were stained for annexin IV (Fig. 2A–D), a strong staining was observed around the yellow lipofuscin granules (Fig. 2A, open arrow). Additionally a punctate longitudinal staining was also evident as shown in the non-failing left ventricle (Fig. 2A, triangles). Also, the cross striation pattern observed in the non-failing atrium (Fig. 2C) was absent in the failing atrium (Fig. 2D), which showed diminished intracellular staining. In summary, a loss of annexin IV organization was evident in both failing tissues, (Fig. 2B and D) when compared with the non-failing tissues (Fig. 2A and C).


Figure 2
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  		Fig. 2 Immunolocalization of annexins IV, V and VI in the non-failing and failing human heart. In all images, the specific fluorescent stain for annexins is green, and the nuclei are red. Yellow autofluorescent granules are lipofuscin. The magnification for all the images is 63x and the scale bar (in image L) represents 20 µm. Cryostat sections of non-failing left ventricle (A, F and I), failing left ventricle (B, F and J), non-failing right atrium (C, G and K) and failing right atrium (D, H and L) were incubated overnight at 4°C with either anti-annexin IV (A–D), anti-annexin V (E–H) antibody or anti-annexin VI (I–L) antibody and then with FITC-labelled secondary antibody. Tissue sections incubated with the secondary antibody alone were negative for the green fluorescence (not shown). Specific localization of each annexin in each tissue type is discussed in the text.

 
Interestingly, although our Western blots showed a possible cross reaction of the anti-annexin IV antibody with the annexin VI protein, in these immunostaining experiments the characteristic plasma membrane staining for annexin VI was not evident when the tissues are incubated with anti-annexin IV antibody, demonstrating no important cross-reactivity in these studies.

Fig. 3 shows a representative Western blot and bar graph of annexin IV protein levels in the left ventricle and right atrium from non-failing, ICM and DCM hearts. There was a statistically significant increase in the amount of annexin IV protein in both ICM and DCM hearts as compared to non-failing hearts. In the right atrium, however, there were no differences in the amount of protein between either DCM and non-failing or ICM and non-failing hearts.


Figure 3
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  		Fig. 3 Representative Western blot analysis for annexin IV and the summarized bar graphs for left ventricle and right atrium from non-failing (N), ischemic (I) and dilated cardiomyopathic (D) human hearts. Western blot analysis was performed on all 18 hearts. For conditions of the Western blot analysis, see Materials and methods. On all blots, 17, 33 and 51 µg of total left ventricle (top) and right atrium (bottom) protein from the same non-failing heart (standard heart) was blotted. For normalization purposes, densitometric units obtained from each heart were divided by the average value of the standard heart from the same blot. Each value represents the mean of three independent determinations. *P<0.05 vs NF.

 
3.2 Annexin V
Longitudinal sections from left ventricle and right atrium stained with anti-annexin V antibody are shown in Fig. 2E–H. In these sections, a clear cross-striated pattern is observed in all tissues (Fig. 2E–H). In the failing ventricle (Fig. 2F) and atrium (Fig. 2H), however, a more disorganized cross-striated pattern is evident, with areas where annexin V is localized as green brilliant dots in the cytosol (brackets in Fig. 2F and H). In addition, staining for annexin V accumulated around the lipofuscin granules and at the intercalated discs (arrowhead in Fig. 2E). Staining for annexin V in the cytosol of non-myocyte cells is also evident in the majority of the sections and is clearly shown in Fig. 2G (bidirectional arrow).

Fig. 4 shows representative Western blot analyses and the corresponding bar graphs for annexin V in ventricle and atrium from failing and non-failing hearts. Results show a significant increase in the protein levels of annexin V in the left ventricle of ICM and DCM as compared to non-failing. In the right atrium, there were no significant changes in the expression of annexin V between non-failing and either group of failing hearts.


Figure 4
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  		Fig. 4 Representative Western blot analysis for annexin V and the summarized bar graphs for left ventricle and right atrium from non-failing (N), ischemic (I) and dilated cardiomyopathic (D) human hearts. Western blot analysis was performed on all 18 hearts. For conditions of the Western blot analysis, see Materials and methods. On all blots 17, 33 and 51 µg of total left ventricle (top) and right atrium (bottom) protein from the same non-failing heart (standard heart) was blotted. For normalization purposes, densitometric units obtained from each heart were divided by the average value of the standard heart from the same blot. Each value represents the mean of three independent determinations. *P<0.05 vs NF.

 
3.3 Annexin VI
Annexin VI (Fig. 2I–L) was localized at the sarcolemma and intercalated disc in the ventricle and atrium from both non-failing (Fig. 2I and K) and failing (Fig. 2J and L) human hearts. However, in the non-failing left ventricle (Fig. 2), a striation pattern was also observed in the majority of the sections (closed arrow). This pattern was absent in the majority of failing left ventricle sections (Fig. 2J). The failing atrium (Fig. 2L) also showed staining in a longitudinal pattern, but this pattern was not observed in the non-failing atrium (Fig. 2K).

A representative Western blot analysis and the corresponding bar graphs for annexin VI in total homogenate of left ventricle and right atrium of failing and non-failing human hearts are shown in Fig. 5. There were no significant differences in the protein levels of annexin VI between non-failing and ICM or non-failing and DCM hearts in either left ventricle or right atrium.


Figure 5
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  		Fig. 5 Representative Western blot analysis for annexin VI and the summarized bar graphs for left ventricle and right atrium from non-failing (N), ischemic (I) and dilated cardiomyopathic (D) human hearts. Western blot analysis was performed on all 18 hearts. For conditions of the Western blot analysis, see Materials and methods. On all blots 17, 33 and 51 µg of total left ventricle (top) and right atrium (bottom) protein from the same non-failing heart (standard heart) was blotted. For normalization purposes, densitometric units obtained from each heart were divided by the average value of the standard heart from the same blot. Each value represents the mean of three independent determinations. A value of P<0.05 was accepted as statistically significant.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
We chose to study left ventricular tissue because of the large contribution of left ventricular performance to overall cardiac output and its impairment during heart failure. Right atrium was studied in preference to left atrium because the human right atrium is trabeculated, facilitating the removal of muscle preparations comparable to those found in the ventricle, and thus more easily compared.

In this study, we have shown that (1) annexin IV, V and VI are localized in distinct cellular compartments within the cardiomyocytes of the human heart and, in some cases, the characteristic intracellular location of these proteins is altered in the failing human heart; (2) annexin IV and V protein levels are up-regulated in the left ventricle but not in the right atrium of failing human hearts; and (3) annexin VI is not significantly altered in the ventricle or atrium of failing human hearts.

It has been established that human heart failure is characterized by contractile changes, which are accompanied by abnormalities in intracellular Ca2+ cycling [36] and alterations in the steady state levels of both mRNA and proteins involved in Ca2+ cycling, (see Ref. [37] for review). Changes in the expression of E–F hand motif Ca2+ binding proteins, such as calmodulin [24] have also been reported. Disorganization of sarcomeres and the cytoskeleton, as well as an abnormal arrangement of contractile proteins, has been reported in human heart failure [38,39]. Differential regulation of both the amount and the location of other proteins involved in Ca2+ cycling, such as the annexins, has also recently been reported in human heart failure [29].

The fact that annexins IV, V and VI immunolocalize in different compartments within cardiomyocytes may suggest a different role for each annexin in the heart. Moreover, the up-regulation of two of these proteins in the left ventricle, the most dysfunctional chamber in the failing heart, along with other reported abnormalities in Ca2+ cycling, supports the hypothesis that annexins may play a role in Ca2+ handling in the human heart.

Annexin IV is one of the most abundant annexins present in many cell types. Neither the function nor the location of the annexin IV protein has been investigated in the heart. However, in epithelial cells, annexin IV is known to be a cytoplasmic marker of the polarization of the cell, either at the basolateral domain [40] or at the apical domain [41] and it has also been shown to modulate a Ca2+ dependent chloride channel in these cells [14]. In the human heart, our results show that annexin IV is localized in the cytoplasm of cardiomyocytes and that in the failing tissues there is a loss of intracellular organization. Annexin IV protein levels are also significantly increased in the ventricle of both DCM and ICM hearts.

Annexin V and VI have been described as the major cardiac annexins [26]. Their exact function, as well as the immunolocalization of annexin V, remain controversial. It has been shown in both rat [42] and guinea pig [43] hearts that annexin V is localized on the intercalated discs and on the sarcolemma of cardiac isomyocytes. Spreca and colleagues found annexin V localized at the sarcolemma, T-tubule system and intercalated discs [44]. Doubell and co-workers described an immunoreactivity at the sarcolemma of atrial muscle, and the intercalated disc of ventricular muscle, as well as a striated pattern in both atrium and ventricle [26]. The same striation pattern was observed later when Wang and colleagues examined double stained rat cardiomyocytes with anti-annexin V and anti-{alpha}-actin by confocal laser scanning microscopy, suggesting that annexin V localized in association with the z-line of rat cardiomyocytes and was associated with the actin filaments [28]. Similarly, in the human heart, our current results show that annexin V immunolocalizes with a cross-striated pattern spaced at approximately 2 µm, suggesting a localization at the z-line. In the failing heart, we show that annexin V tends to cluster in the cytosol of cardiomyocytes with a lack of organization. These results are consistent with the findings of Hein and co-workers [39] whose immunohistochemistry experiments show changes in both the amount and organization of titin, myosin and thin filament complex in human hearts.

The localization of annexin V at the z-line, and also the up-regulation of annexin V protein in the left ventricle, are consistent with a role for annexin V in altered Ca2+ handling in the human heart during failure. The up-regulation of annexin V in DCM and ICM human hearts agrees with the recent results of Song and colleagues, who showed an increase in both the mRNA and protein levels for annexin V in the failing human heart [29].

The immunolocalization of annexin VI is less controversial. It has been shown to be present at the sarcolemma and the intercalated disc of rat and porcine cardiomyocytes [26,43]. In the human heart, our results also show a sarcolemmal and intercalated disc distribution of annexin VI in both atrium and ventricle. Functionally, Sobota and co-workers reported that annexin VI could reverse the inhibition of Na+/Ca+2 exchange activity of cardiac sarcolemmal vesicles [18], which is consistent with its localization. This function may be particularly relevant in human heart failure, where a number of studies have demonstrated changes in the amount or function of the Na+/Ca2+ exchanger [5,45].

Evidence for the physiological importance of annexin VI in the heart comes from studies of Gunteski-Hamblin and colleagues, who overexpressed annexin VI in the mouse heart and reported that the cells isolated from these hearts showed a frequency-dependent reduction in the percent shortening as well as decreased rates of contraction and relaxation. The transgenic animals had lower basal levels of intracellular free Ca2+ and a reduced rise in free Ca2+ following depolarization. After contraction, intracellular free Ca2+ returned to basal levels faster in transgenic cells than in those taken from control animals [19]. The relative importance of annexin VI in regulating Ca2+ levels and contractility may be exaggerated in the failing heart, where these processes are altered.

Song and co-workers described a decrease in the protein level, as well as the mRNA level, for annexin VI in failing human hearts compared with non-failing human hearts [29]. Our results show no significant difference in the protein levels of annexin VI between DCM or ICM hearts and non-failing human hearts, although it is worth mentioning that levels in the DCM hearts showed a trend towards a decrease, while in the ICM hearts the trend was an increase in our study. The reason for the difference in annexin VI results between our study and the study of Song and co-workers is not clear. We speculate that differences in the human population used for the studies, particularly the non-failing patient population, may partially explain this discrepancy. Clarification of this issue awaits further study.

In conclusion, the differential subcellular location of annexins and their close proximity with the proteins involved in the handling of Ca2+, as well as changes in their cellular organization and the up-regulation of two of these proteins in the failing heart, may suggest a role for annexins in the regulation of Ca2+ cycling in the heart. Further studies are needed to clarify the functional implications of these findings.

Time for primary review 26 days.


   Acknowledgements
 
The authors gratefully acknowledge the assistance of the Heart Transplant Teams and the Department of Pathology at the Cleveland Clinic Foundation for help in obtaining failing human heart tissue, as well as Life Banc of Northeast Ohio for help in obtaining non-failing human heart tissue. We also thank Dr. Judith Drazba for assistance with confocal microscopy and Sam Fratantonio for tissue sectioning. The work described in this manuscript was supported by NIH HL49929 and National AHA 95007700 and 95-261/EI.


   Notes
 
* Supported by NIH HL49929 and National AHA 95007700 and 95-261/EI. Back

1 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Regulatory Biology at Cleveland State University. Back


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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