Heart failure is a clinical syndrome characterized by the inability of the heart to produce an adequate cardiac output [19
]. A key contributor to the dysfunction may be depressed myocardial contractility [20
]. Studies that have examined the function of cardiac myocytes (CM) isolated from failing hearts suggest that a defect in CM contractility may contribute to the overall cardiac dysfunction [22
]. The responsible agents or mechanisms, however, remain incompletely characterized.
Inflammatory molecules may be agents of myocardial depression in heart failure. The hypothesis that inflammation plays a role in cardiac dysfunction was first suggested from animal [23
] and human studies of sepsis (reviewed by van der Poll et al
]). Sepsis-associated cardiac dysfunction was thought to be mediated, in part, by circulating or “humoral” myocardial depressants that were subsequently identified as predominantly inflammatory cytokines (including tumor necrosis factor-1alpha (TNF-1α), interleukin-1beta, (IL-1β) [25
], IL-6 [26
], IL-2 [27
] and interferon-gamma (IFN-γ)). These ideas were supported by numerous animal [28
] and by in vitro studies [25
]. However, clinical trials (reviewed by Anker et al
]), that used “targeted” approaches to neutralize cytokines, such as TNF, were unsuccessful in treating patients with moderate to advanced heart failure.
Chemokines are a distinct class of inflammatory molecules that are also elevated in heart failure. This class of receptors is present on the myocardium and vascular smooth muscle cell types and is capable of mediating important biological processes in a manner that is independent of inflammatory cells [34
]. Recently, several studies have demonstrated that circulating chemokine levels, including that of CXCL12, are elevated in animals and humans with cardiac dysfunction [35
]. Therefore, chemokines have been proposed as mediators of cardiac dysfunction. However, the mechanism of chemokine-mediated cardiac dysfunction is unknown.
We hypothesized that the role of chemokines and their receptors in the cardiac response to inflammation may be quite distinct from that of cytokines' given the wide degree of structural, functional and regulatory differences between the two classes of molecules. Therefore, there is a rationale for assuming that treatment outcomes for cytokine-directed therapies of heart failure may not be predictive of chemokine receptor-directed modalities. Damas et al.
] demonstrated that a variety of chemokine receptors was increased in the failing human myocardium compared to non-failing specimens. Although the CM is the predominant cell type in cardiac tissue, a potential drawback of this study is that the entire ventricle or atrium was processed and used for RNA protection assay analysis so that the relative contribution of each resident cell type to the elevated chemokine receptor levels could not be determined. Seino et al
] demonstrated that CCL2 (monocyte chemoattractant protein (MCP-1)) RNA levels are detectable in human myocardial biopsies and in TNF-α1-treated neonatal rat CM. These studies postulated that the mechanisms for chemokine effects on myocardial function were predominantly paracrine responses to circulating leukocytes [7
]. Our study demonstrates that the chemokine CXCL12 can directly depress contractility of the myocardium.
In the present study, normal PM exposed to CXCL12 demonstrated a blunted response to Ca2+
stimulation. The concentrations of CXCL12 used in the experiments are similar to those used in measuring the physiologic effects of CXCL12 in a variety of cell types such as epithelial cells [39
], lymphocytes [41
] and neurons [42
]. Histological examination of the PM demonstrated a paucity of inflammatory cells. Therefore, it was unlikely that inflammatory cells mediated the effect of CXCL12 on PM contractility. However, because the papillary muscle is composed of heterogeneous cell types, the effects of CXCL12 could not be ascribed to one particular cell type based on studies employing multi-cellular preparations.
The blunted Ca2+
response of papillary muscles is explained in part by a direct effect of CXCL12 on CM. CM exposed to CXCL12 demonstrated a significantly decreased shortening in response to both Ca2+
and ISO stimulation at higher concentrations. At lower concentrations of Ca2+
(1 mM and 2 mM) and ISO (0.001 μM) CXCL12 exposure did not cause decreased CM shortening. This was consistent with the observation that whereas the failing myocardium shows normal baseline function, the contractile reserve is diminished [43
]. The lack of CXCL12 effect on CM shortening at normal
and low ISO concentration imply that in patients with normal left ventricular function, CXL12 may not have a direct negative effect on contractility. However, in conditions such as heart failure where there is sympathetic activation, it is possible that the presence of CXCL12 induces a negative inotropic effect.
CXCL12 imparts its effects on CM through interaction with its exclusive receptor CXCR4. A highly specific CXCR4 antagonist, AMD3100, abrogated the depressed contractile response to Ca2+, and overexpression of CXCR4 by adenoviral infection of CM depressed the contractile response to Ca2+ treatment even further in the presence of CXCL12. Ligand-induced receptor internalization is a general regulatory mechanism in chemokine signaling. During Ca2+ stimulation, we observed that CM returned to normal contractile response after prolonged CXCL12 exposure, which is consistent with receptor desensitization. These observations demonstrate that the effect of CXCL12 is mediated through its interaction with CXCR4.
Immunofluorescent microscopy showed that there was a significant amount of CXCR4 on or near the surface of human and rat adult CM. The expression appeared to be linear and the receptors in certain sections appeared to be clustered, a finding consistent with reports of CXCR4 expression in other cell types [44
]. Further studies are ongoing to determine the factors that regulate CXCR4 surface expression on CM. The immunohistochemical studies of CXCR4 on human CM demonstrated that CXCR4 expression on CM is not limited to murine species and therefore may be relevant to human diseases.
To determine the possible mechanisms by which CXCR4 activation decreases myocardial function, we examined Ca2+
mobilization within CM when exposed to CXCL12. During Ca2+
stimulation peak, Ca2+
transients were decreased in CM exposed to CXCL12 at supraphysiologic concentrations of (4–8 mM), but not at basal levels (1–2 mM).
CXCL12 may modulate Ca2+
transients by altering L-type Ca2+
channel activity in response to either calcium or an adrenergic challenge.
Patch clamp studies measuring L-type Ca2+
channel activity of individual CM demonstrated that CXCL12 decreased Ca2+
currents induced by either β-adrenergic with ISO or FSK stimulation but CXCR4 activation did not have a significant effect on basal activity of the Ca2+ currents.
Together, these data suggested both an upstream effect of CXCR4 on L-type Ca2+
channels as well as a downstream effect that appears to be unrelated to β-adrenergic receptor stimulation. This is consistent with the findings of others wherein treatment of colonic epithelial cells with CXCL12 inhibited cAMP response only in the presence of FSK stimulation [45
]. It appears that an important function of CXCR4 is to modulate cAMP-mediated events. In CM, this may have profound physiologic implications such as the modulation of contractility as demonstrated in the present studies.
A novel and exciting potential methodology to reverse myocardial remodeling associated with myocardial infarction [46
] is regeneration of myocardium using bone marrow (BM)-derived mesenchymal cells, endothelial progenitor cells and endogenous cells [47
]. Given its essential role in BM homing and recruitment [49
], CXCL12 is a candidate to promote stem cells recruitment to the heart. Because CXCR4 may negatively impact myocardial function, strategies [52
] that primarily employ up-regulation of CXCL12 may need to be examined more closely. Our results would caution that a therapeutic approach whereby myocardial CXCL12 is increased, in an effort to recruit circulating or endogenous stem cells in the setting of an ischemic injury, may result in a significant decrease in contractility.
In this study we have shown, for the first time to our knowledge, that CXCR4 is functional on CM. CXCR4 activation can directly inhibit myocardial contractile response to increases in Ca2+ and ISO concentrations through modulation of L-type Ca2+ channel activity. Manipulation of CXCR4 activity may serve as a new target in the treatment of cardiac dysfunction. Additionally, the discovery of functional chemokine receptors on CM may have broad implications on preexisting chemokine/chemokine receptor based stem cell therapies for cardiomyopathies.