Endothelin-1 (ET-1), a potent vasocontractile peptide produced by endothelial cells, is also produced by cardiac myocytes. ET-1 induces cardiac hypertrophy and cellular injury of cardiac myocytes in addition to its potent positive inotropic and chronotropic actions. ET-1 has arrhythmogenic actions on the heart. It has been reported that plasma ET-1 concentrations are elevated in heart failure in humans and in experimental animal models. These studies suggest the involvement of the ET-1 pathway in the pathophysiology of heart failure.

The endothelin receptor antagonist, BQ-123, was shown to greatly improve survival in rats with heart failure by Miyauchi and colleagues at the University of Tsukuba. An endothelin receptor antagonist was also shown by this group to improve the decrease in expression of functional molecular markers mRNA levels of the ryanodine receptor and the sarcoplasmic reticulum CA2+-ATPase. This suggests that one mechanism for favorable effect of the endothelin receptor antagonist is amelioration of the decrease in these markers and amelioration of the calcium overload of myocardial cells.

This group has also shown that chronic treatment with an endothelin receptor antagonist normalized the increase in ACE mRNA in heart failure rats, and the increase in AT1 receptor mRNA in the failing heart of rats with heart failure due to myocardial infarction. Therefore, suppression of the renin angiotensin system (RAS) may be one mechanism by which the endothelin receptor antagonist exerts its beneficial effect.

The cross-talk between the endothelin and RAS systems was studied by this group using transgenic hypertensive mice with hypertrophy and overexpressing angiotensin II, which have both human renin and human angiotensinogen due to the mating process. Because ET-I is augmented by angiotensin II in vitro, mRNA in the hearts of the transgenic hypertensive rats was measured. ET-1 mRNA was greatly enhanced in these mice hearts in vivo. Chronic treatment with an endothelin receptor blocker greatly ameliorated the hypertrophy seen in the control mice. Therefore, in renin angiotensin overexpressing mice, an endothelin receptor antagonist greatly ameliorated the cardiac hypertrophy. Thus, it can be speculated, stated Miyauchi, that angiotensin II has a direct effect on cardiac myocytes and induces endothelin, which causes cardiac hypertrophy.

Combined treatment with an endothelin receptor antagonist and an ACE inhibitor improved survival to about 90% in hamsters with heart failure, compared to about a 45-50% survival with an endothelin receptor alone, about a 35% survival with an ACE inhibitor, and about a 5% survival with no treatment. This shows that both the endothelin and RAS systems contribute independently to the progression of heart failure and that there is cross talk between these two systems.

Miyauchi and colleagues hypothesize that in the failing heart, impaired myocardial energy metabolism occurs. The impairment of beta oxidation of fatty acids causes activation of the glycolytic system, and this switch in metabolic energy induces increased cardiac gene expression of ET-1. Experiments they subsequently conducted supported this hypothesis.

An increase in oxygen consumption by cardiac myocytes and a switch in the principle ATP energy source from mitrochondrial fatty acid oxidation to a glycolytic system are involved in the pathophysiology of heart failure. Since ATP production per oxygen consumption by glycolysis is greater than that by mitochondrial oxidation in the failing heart, this alteration in energy metabolism may have a compensatory aspect for the failing heart as an adaptation. Hypoxia-inducible factor (HIF)-1, a transcriptional factor, is known to be involved via the increased expression of glycolytic enzymes. HIF-1 activates the gene expression of glycolytic enzymes, which are activated as compensation for mitochondrial oxidation of fatty acids in the failing heart. Miyauchi and colleagues identified by sequence analysis of the 5’-flanking promoter region of ET-1 an HIF-1 recognition site in this region. Therefore, they hypothesized that HIF-1 is involved in the increase in the myocardial expression of the ET-1 gene in heart failure.

Work by Miyauchi and colleagues showed that HIF-1 transcriptionally activates ET-1 gene expression by direct interaction with the predicted DNA binding site in the 5’- promoter region of the ET-1 gene in rat cardiomyocytes. HIF-1 mRNA and ET-1 mRNA in the failing heart increased during the aggravation of heart failure in rats with myocardial infarction and hamsters with cardiomyopathy. In rat cultured myocytes treated with a mitochondrial inhibitor, HIF-1 mRNA and ET-1 mRNA was markedly increased with activated glycolysis. Furthermore, antisense oligonucleotide for HIF-1 greatly inhibited this expression of ET-1 in cardiomyocytes.

Miyauchi concluded that ET-1 is an important aggravating substance in heart failure. Various factors are involved in the increase in ET-1 gene expression in the failing heart. Their recent data has revealed a novel molecular mechanisms for the upregulation of cardiac ET-1 in heart failure in which impairment of cardiac energy metabolism is involved in an increase in ET-1 expression through HIF-1, suggesting that induction of HIF-1 to stimulate glycolysis as an adaptation against impaired energy metabolism alternatively causes an elevation of cardiac ET-1 expression as a maladaptation, which leads to aggravation of heart failure.

The role of the natriuretic peptide system in the basal condition was evaluated in the double knockout mice model of GC-A, a receptor for natriuretic peptides, and type I angiotensin II (Ang II) type receptor genes by Saito and colleagues at the University of Kyoto. Data from GC-A knockout mice clearly indicate that the ANP-GC-A system functionally antagonized humoral factors or pathways that lead to hypertension, left ventricular hypertrophy (LVH) and cardiac fibrosis.

The actions of ANP and BNP are opposite those of Ang II at every site of action. Thus to elucidate the pathways for the functional antagonism of these peptides, they generated a ANP-GC-A knockout mice. Systolic blood pressure (SBP) was significantly higher in the GC-A knockout mice than in the wild mice, consistent with previous reports, while the SBP was significantly lower in the AT1a knockout mice than in the wild type. The SBP was significantly lower than that in the GC-A knockout mice but significantly higher than that in the AT1a knockout mice. The degree of reduction in SBP between the wild type and the AT1a knockout mice was similar to the degree of reduction from the GC-A knockout to the double knockout mice—indicating that the genetic blockade of the AT1a receptor similarly decreases the SBP in the wild and GC-A knockout mice. Thus, it is unlikely that Ang II contributed to blood pressure elevation in the GC-A knockout mice. Their work also showed that the genetic blockade of AT1a was associated with a greater reduction in the ratio of LV weight to body weight in the double knockout mice, compared to the GC-A knockout and the AT1a knockout mice models. Also they showed that the GC-A knockout mice had more LV interstitial fibrosis than in the wild type or the AT1a knockout mice. Less LV fibrosis was observed in the double knockout mice than in the GC-A knockout mice.

TGF-beta, collagen I and collagen III mRNA expression, genes known to be responsible for fibrosis, was completely blocked in the double knockout mice compared to the AT1a knockout mice. The percent inhibition of SBP was similar in the AT1a and double knockout mice. The percent inhibition of LVW/BW and fibrosis was significantly greater in the double knockout mice than in the AT1a knockout mice. These results indicate that the endogenous ANP-GC-A system plays an important role in the functional inhibition of endogenous Ang-II-mediated hypertrophy and fibrosis. However, the ANP-GC-A system functionally blocks some pressor pathways other than Ang II in the basal condition.

New roles for natriuretic peptides in the setting of acute myocardial infarction using GC-A knockout mice were also investigated by this group. Plasma BNP is markedly elevated in patients who undergo PCI after AMI. To determine the role of BNP or the ANP system in the acute phase of MI, they developed a mouse model of ischemia reperfusion using the GC-A knockout mice. The ratio of the area of the infarct area to the area at risk was about 60% in the wild type and about 45% in the GC-A knockout mice. The infarct size was significantly decreased by about 25% in the GC-A knockout mice at 2 days after reperfusion.

PMN infiltration in the GC-A knockout mice was much smaller than in the wild type mice at 6 hours and 2 days after reperfusion. Increasing evidence shows that the PMN infiltration into the infarct region follows three steps, rolling migration, firm attachment, and transmigration. The upregulation of P-selectin in the endothelial cells play an important role in binding to the PMN.

To understand the mechanism of increased infiltration of PMN in the infarct region of the GC-A knockout mice, they examined the endothelial expression of P-selectin after ischemia reperfusion. P-selectin was abundantly observed in the vessels in the perinecrotic region of the viable myocardium near the infarction. In contrast, few P-selectin cells are seen in the GC-A knockout mice.

Activation of NF-kB, transcriptional factor that upregulates the selectin gene in the endothelial cells, is much weaker in the GC-A knockout mice than in the wild type mice. P-selectin expression was increasingly upregulated in human endothelial cells by the addition of hydrogen peroxide, hydrogen peroxide plus ANP, and even more with hydrogen peroxide plus ANP plus an antagonist for GC-A.

Based on the data, these investigators proposed a new role for GC-A in ischemia reperfusion. Radical oxygen species generated by ischemia reperfusion phosphorylate IkB, which leads the activation of NF-kB. Activated NF-kB leads to the transcription of P-selectin and then to PMN infiltration. GC-A or cyclic GMP potentially regulates the ROS-induced NF-kB activation. These findings provide a new insight into the role of the natriuretic peptide system in ischemia reperfusion (Figure 1).

A decrease either in the activity or the protein expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase is thought to be a major determinant in the pathogenesis of cardiac dysfunction in heart failure.

A novel mechanism of cardiac dysfunction elucidated by assessing the functional interaction of the FK 506-binding protein (FKBP12.6) with the cardiac ryanodine receptor (RyR) in a canine model of pacing-induced heart failure has been reported by Yano and colleagues at Yamaguchi University and Tohoku University Graduate School of Medicine. They showed that in heart failure the stoichiometry of FK binding protein (FKBP) per RyR was decreased. This partial loss of RyR-bound FKBP12.6 seems to induce an instability in the properties of the channel through a protein conformational change. This leads to a prominent Ca2+ leak, which can cause Ca2+ overload and hence diastolic and systolic dysfunction.

It has been recently demonstrated that FKBP are tightly coupled with the RyR with a stoichiometry of 1 RyR to 4 FKBP. Although FKBP appears to have a channel stabilizing effect in skeletal muscle, controversy remains regarding the role of cardiac FKBP on the function of RyR. Thus, they assessed the role of the interaction between FKBP and RyR in the pathogenesis of heart failure.

Heart failure was created by chronic rapid right ventricular pacing at a rate of 250 bpm for 3 weeks in the canine model. After hemodynamic assessment, the LV was isolated and the SR vesicles were purified. The calcium leak from its initiation to its saturation was measured in the presence of various concentrations of FK506. It is known that FK506 specifically binds to the FKBP and then dissociates FKBP from the RyR.

The hemodynamics showed that the LV, diastolic and end systolic diameters and the LV end diastolic pressure were increased in the heart failure model. There was a decrease in fractional shortening. The +dP/dt pressure of the LV pressure was decreased.

In normal SR, FK506 caused a dose-dependent calcium leak that was similar to the significant conformational change in RyR. In contrast, in the failing SR, a prominent calcium leak was observed even in the absence of FK506, and FK506 produced little or no further increase in calcium leak and only a slight conformation change in RyR.

Calcium release assay using stopped-flow apparatus was performed. In normal SR vesicles, the addition of FK506 decreased the rate of calcium release. However, in heart failure, FK506 did not decrease the rate of calcium release, which was already decreased compared to in heart failure. These data indicate that some of the FKBP dissociated by FK506 is already partially lost in heart failure.

A polylysine-induced increase in ryanodine receptor binding means deactivation of the ryanodine receptor. In normal conditions, FK506 decreased the polarizing induced enhancement of the ryanodine binding in a dose-dependent fashion. However, in heart failure, FK506 had no significant effect on polylysine-induced increase in ryanodine receptor binding. In heart failure, the Bmax of the FK506 was markedly decreased. However, the Kd was unchanged. The stoichiometry of FKBP per RyR was 1:3.6 in the normal heart, compared to 1:1.6 in heart failure. The protein expression of FKBP12.6 was significantly decreased in heart failure compared to normal SR vesicles (p<0.05; Figure 2).

Yano concluded that in tachycardia-induced heart failure, the rate of calcium release through RyR was decreased in association with an abnormal calcium leak through the RyR. This abnormality within this protein is presumably caused by a partial loss of RyR-bound FKBP12.6 and the resultant conformation change in RyR. This abnormal channel gating in RyR might possibly cause calcium overload and consequent diastolic and systolic dysfunction.

Heparin binding epidermal growth factor (HB-EGF) was purified from macrophage-like cells U937 by Takashima and colleagues at Osaka University. HB-EGF is a mitogen for fibroblasts and vascular smooth muscle cells (VSMC) but not for endothelial cells. HB-EGF is involved in the pathogenesis of atherosclerosis. Neonatal and adult cardiac muscle cells respond to both neurohumoral and mechanical growth stimuli with a marked increase in HB-EGF mRNA. The expression and protein synthesis of HB-EGF was enhanced in the LV of spontaneous hypertensive rats.

They investigated whether HB-EGF shed by metalloproteinases plays an important role in the development of cardiac hypertrophy. They hypothesized: That HB-EGF is involved in cardiac hypertrophy. HB-EGF plays an important role in the cardiac hypertrophic signaling by catecholamine, angiotensin II and endothelin-1. The cellular mechanisms by which these factors are linked with HB-EGF are attributable to the shedding of HB-EGF via metalloproteinases. Therefore, a metalloproteinase inhibitor attenuates cardiac hypertrophy in vivo.

HB-EGF can phosphorylate the EGF receptor, and induce ERK activation and cardiac hypertrophy, as shown by this group. Further, the G-protein coupled receptor (GPCR) agonists such as phenylephrine, angiotensin II, and endothelin-1 can stimulate HB-EGF release, and the EGF receptor phosphorylation can occur extracellularly, induce ERK activation and cardiac hypertrophy.

The metalloproteinase inhibitor KB-R7785 can block the GPCR agonist-induced hypertrophic stimulation. They also showed that KB-R7785 can block EGFR phosphorylation in rat neonatal cardiomyocytes. In a diabetic mouse model of cardiac hypertrophy, KB-R7785 inhibited the shedding of HB-EGF. In another experiment, they showed that KB-R7785 attenuated cardiac hypertrophy induced by aortic binding. LV thickness was also attenuated by KB-R7785. The metalloprotienase inhibitor attenuates cardiac hypertrophy in either diabetic or pressure overloaded mice.
Takashima concluded that HB-EGF plays an important role in the development of cardiac hypertrophy by the shedding of HB-EGF via metalloproteinase. The present results hint that the inhibition of the novel pathway of metalloproteinase-HB-EGF signaling in cardiac hypertrophy may be therapeutic in cardiac hypertrophy.