Regulation of cardiac responses to increased load

Role of endothelin-1, angiotensin II and collagen XV

Jarkko Piuhola

Department of Pharmacology and Toxicology, University of Oulu
Biocenter Oulu, University of Oulu

Abstract

Chronic overload of the heart is the major cause of left ventricular hypertrophy (LVH) and eventually heart failure. It is generally accepted that autocrine/paracrine factors, such as angiotensin II (Ang II) and endothelin-1 (ET-1) contribute to the development of LVH. Cardiac hypertrophy and failure are characterized by attenuated responsiveness to β- adrenergic stimulation and accumulation of collagenous material to the left ventricular wall. The present study aimed to characterize the roles of ET-1 and Ang II in the regulation of cardiac function. The role of the plasmamembrane Ca2+-ATPase (PMCA) in ET-1 induced cardiac responses and the role of type XV collagen in cardiac function were also studied.

Both ET-1 infusion and mechanical loading were able to induce positive inotropic effect and induction of early response genes in isolated perfused hearts. ET-1 also induced strong vasoconstriction. Cardiomyocyte-specific PMCA overexpression inhibited the ET-1 induced hypertrophic response, while inotropic response remained unaltered. ET-1 was found to induce release of adrenomedullin (AM), a potent vasorelaxing and inotropic peptide. Infusion of AM antagonized the vasoconstrictive effect of ET-1 independently of nitric oxide. In hypertrophied rat hearts ET-1 was found to contribute significantly to the Frank-Starling response, a fundamental mechanism regulating contractile performance of the heart. In mice hearts, ET-1 was found to play a dual role in load induced elevation of contractile strength: ETA receptors mediated an increase, while ETB receptors mediated an inhibitory effect on contrcatile force. Ang II was not contributing to the contractile response to load in either rat or mice hearts. Blunted response to β-adrenergic stimulus and increased vulnerability as a result of exercise was observed in mice lacking collagen XV.

In conclusion, the present results underscore the importance of the local factors, especially ET-1, in regulation of cardiac function, not only in terms of hypertrophic but also in terms of contractile response to load. The results also suggest a role for PMCA in regulation of cardiac function. Lack of type XV collagen was found to result in cardiac dysfunction with many features similar to those of early heart failure.

Dedication

To Päivi

Table of Contents
Acknowledgements
Abbreviations
List of original papers
1. Introduction
2. Review of the literature
2.1. Regulation of cardiac contractile function
2.1.1. Excitation-contraction coupling
2.1.2. The Frank-Starling mechanism
2.1.3. The force-frequency relationship
2.1.4. The adrenergic system
2.1.5. Circulating hormones
2.2. Autocrine/paracrine factors
2.2.1. Endothelins
2.2.2. Angiotensin II
2.2.3. Adrenomedullin
2.2.4. Nitric oxide
2.2.5. Other paracrine mediators
2.3. Changes in cardiac gene expression and structure in response to increased load
2.3.1. Mechanotransduction
2.3.2. Cardiac gene expression response to load
2.4. Natriuretic peptide system
2.5. Cardiac extracellular matrix
2.6. Genetically engineered animal models in cardiovascular research
3. Aims of the research
4. Materials and methods
4.1. Materials
4.2. Experimental animals
4.3. Isolated perfused heart preparations (I-V)
4.4. Exercise experiment (V)
4.5. Experimental protocols
4.6. Isolation and analysis of cytoplasmic RNA (I-III, V)
4.7. Radioimmunoassays (I- III)
4.8. Cyclic AMP measurements (V)
4.9. Analysis of markers for cardiac injury (V)
4.9.1. TUNEL-staining
4.9.2. Preparation of samples for biochemical assays
4.9.3. ProMMP-2 activity
4.9.4. β -glucuronidase activity
4.10. Histology (IV, V)
4.10.1. Light microscopy
4.10.2. Electron microscopy
4.11. Statistical analysis
5. Results
5.1. Cardiac overexpression of the plasma membrane Ca2+-ATPase (I)
5.1.1. Effects on baseline cardiac function
5.1.2. Effects on responses to endothelin-1
5.1.3. Effects on responses to mechanical load
5.2. Effects of adrenomedullin on endothelin-1 induced coronary vasoconstriction (II)
5.3. Frank-Starling response in the hypertrophied double transgenic rat hearts (III)
5.3.1. Baseline characteristics of the double transgenic rats harboring human renin and angiotensinogen genes
5.3.2. Effects of loading
5.3.3. Effects of bosentan and CV-11974 on the Frank-Starling response
5.4. Mechanical load induced responses in mice hearts (IV)
5.4.1. Effects of atrial and ventricular loading
5.4.2. Roles of endothelin-1 and ETA and ETB receptors
5.5. Role of type XV collagen in cardiac structure and function (V)
5.5.1. Effects of isoproterenol on cardiac function
5.5.2. Changes in cardiac stress responses in collagen XV deficient mice
6. Discussion
6.1. Modulation of endothelin-1 induced cardiac effects by plasma membrane Ca2+-ATPase overexpression
6.2. Adrenomedullin in regulation of coronary vascular tone
6.3. Endothelin-1 in regulation of cardiac contractile function
6.4. Distinct roles of ETA and ETB receptors in mice hearts
6.5. Collagen XV and cardiovascular structure and function
7. Summary and conclusions
References
List of Tables
1. Function of ET receptors in different cell types of cardiovascular system.
2. Summary of ventricular genes induced in response to cardiac load.
3. Genetically engineered mice with alterations in the natriuretic peptide system
4. Summary of the experimental protocols.
5. Primer and probe sequences used for mRNA quantitation
6. Contractile function in NTG and dTG rat hearts.
List of Figures
1. Calcium fluxes during cardiac cycle. Gray boxes with per cent values indicate proportion of Ca2+ removal during diastole by the respective mechanism in human and rabbit hearts. IP3R, IP3 receptor; L-CaCh, L-type Ca2+ channel; NHE, Na+-H+ exchanger; NCX, Na+-Ca2+ exchanger; PLB, phospholamban; PMCA, plasma membrane calmodulin-dependent Ca2+ ATPase; RyR, Ryanodine receptor; SERCA, sarcoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; TnC, troponin C. Modified from Bers 2000.
2. Schematic presentation of biosynthesis and structure of ET-1. aa, amino acid; Ang II, angiotensin II; ANP, atrial natriuretic peptide; ECE, endothelin converting enzyme; ET-1, endothelin-1; IL-1, interleukin-1; NO, nitric oxide. Modified from Giannessi et al. 2001.
3. An overview of cellular events leading to enhanced contractility and hypertrophic response after exposure to ET-1 in cardiomyocytes. AC, adenylyl cyclase; DAG, diacylglycerol; IP3, inositol-1,4,5-triphosphate; MAP kinase, mitogen activated protein kinase; NCX, Na+-Ca2+ exchanger; NHE, Na+-H+ exchanger; PKA, protein kinase A; PKC , protein kinase C; PLC, phospholipase C. Ballard & Schaffer 1996, Shigekawa & Iwamoto 2001 and Takeuchi et al. 2001.
4. A simplified summary of cardiac response to load leading to enhanced contractile force and hypertrophy. Modified from Crozatier 1996 and Perez et al. 2001.
5. A schematic presentation of basement membrane structure surrounding cardiac cells. Modified from Eklund et al. 2000, Sasaki et al. 2000 and Towbin & Bowles 2001.
6. Panel A. Changes in BNP gene expression in response to 2-hour stimulation with ET-1 or mechanical load (elevated coronary flow) in NTG and PMCA rat hearts. Panel B Changes in AM and c-fos gene expression in response to 2-hour stimulation with ET-1 in NTG and PMCA rat hearts. Results are means  SEM. *P < 0.05, †P < 0.01, and ‡P < 0.001 vs. control (Student"s t-test)
7. Immunoreactive (ir)-BNP and ir-AM secretion during ET-1 infusion in NTG and PMCA rat hearts. Solid line, NTG rats; dashed line, PMCA rats. open box, NTG control; open circle, NTG ET-1; black box, PMCA control; black circle, PMCA ET-1. Data are means  SEM. *P < 0.05 vs. NTG control and vs. PMCA ET-1; † P < 0.005 vs. SD control (repeated-measures ANOVA).
8. Attenuation of the vasoconstrictor effect of ET-1 by AM in isolated rat hearts. After a control period, ET-1 (0.08 and 1 nmol/l) (A) and/or AM (0.03 and 1 nmol/l) was added to the perfusion fluid in the presence or absence of nitric oxide synthase inhibitor L-NAME (300 M) (B) for 30 min. Results are expressed as % change vs. baseline values. Each point is the mean  SEM from 6-7 separate experiments on different isolated rat hearts. *P < 0.001 AM vs. vehicle; P < 0.001 ET-1 + L-NAME vs. ET-1; P < 0.01 ET-1 + L-NAME + AM vs. ET-1 + L-NAME; §P < 0.05 ET-1 vs. vehicle; P < 0.001 AM vs. vehicle; ¶ P < 0.001 ET-1 + L-NAME vs. ET-1; #P < 0.001 ET-1 + L-NAME + AM vs. ET-1 + L-NAME by two-way analysis of variance for repeated measurements. Note the different scale of pressure change between A and B.
9. Plots showing the developed pressure (DP), maximal positive and negative derivative of intraventricular pressure (+dP/dtmax, and -dP/dtmin) and left ventricular end diastolic pressure (LVEDP) in NTG and dTG rat hearts during stepwise increment of intraventricular balloon volume. NTG and dTG differ significantly in all parameters (P < 0.01) (one-way ANOVA followed by Student-Newman-Keul´s post hoc test).
10. Panel A. Plots showing changes in Frank-Starling responses as measured by developed pressure (DP) and maximal positive derivative of intraventricular pressure (+dP/dtmax) in response to treatment with ETA/B antagonist bosentan (1 ìM), AT1 antagonist CV-11974 (10 nM) or vehicle in NTG and dTG rat hearts. * P < 0.05 vs. respective vehicle infused group. Panel B. Plots showing diastolic properties as measured by LVEDP in response to bosentan, CV-11974 or vehicle in NTG and dTG rat hearts during the stepwise increment in intraventricular balloon volume. P = NS vs. respective vehicle group (one-way ANOVA followed by Student-Newman-Keul´s post hoc test).
11. Panel A. Coronary artery branch exposed to elevated arterial perfusion pressure (170 mmHg). A large coronary artery branch with preserved endothelial cell lining. Panel B. Coronary artery branch exposed to saponin (100 g/L). The arterial lumen is filled with detached endothelial cells (vWF, hematoxylin counterstain).
12. Perfusion pressure during different treatments in isolated mice hearts perfused with control flow rate of 2 mL/min or loaded with flow rate of 5 mL/min. Each point is the mean  SEM from 6-7 separate experiments on different isolated hearts. *P < 0.05 (ANOVA followed by Student-Newman-Keul´s post hoc test).
13. Panel A. Effect of ET- and Ang II receptor antagonists on DT in loaded mice hearts during 30-minutes loading with elevated coronary flow rate. *P < 0.05 vs. vehicle (ANOVA). Panel B. Maximal DT elevation during loading in different groups during loading. B, bosentan; C, CV-11974; *P < 0.05 vs. vehicle (Student’s t-test).
14. Responses to low doses of isoproterenol at six months (Panel A) and one year age (Panel B) in isolated perfused hearts of Col15a1-/- mice (Closed circles) and wild-typed littermates (Open boxes) (n = 7-11). The baseline developed pressure was 31.7  5.0 and 27.3  5.0 mmHg for the 6-month-old control and null mice, respectively, and 20.2  3.4 and 20.8  2.0 mmHg for the 1-year-old mice (mean  SEM). DP; developed pressure. *P < 0.05 **P < 0.01 (one-way ANOVA followed by Student-Newman-Keul´s post hoc test).
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