CXCR4 modulates contractility in adult cardiac myocytes

Abstract

The inflammatory response is critical to the development and progression of heart failure. Chemokines and their receptors are a distinct class of inflammatory modulators that may play a role in mediating myocardial dysfunction in heart failure. Levels of the chemokine CXCL12, also known as stromal cell-derived factor (SDF), and its receptor, CXCR4, are elevated in patients with heart failure, and we undertook this study to determine whether this chemokine system can directly affect cardiac function in the absence of leukocytes. Murine papillary muscles and adult rat cardiac myocytes treated with CXCL12, the only identified ligand of CXCR4, demonstrate blunted inotropic responses to physiologic concentrations of calcium. The negative inotropic effects on cardiac myocytes are accompanied by a proportional diminution of calcium transients. The effects are abrogated by AMD3100, a specific CXCR4 inhibitor. Overexpression of the receptor through adenoviral infection with a CXCR4 construct accentuates the negative inotropic effects of CXCL12 on cardiac myocytes during calcium stimulation. CXCR4 activation also attenuates beta-adrenergic-mediated increases in calcium mobilization and fractional shortening in cardiac myocytes. In electrophysiologic studies, CXCL12 decreases forskolin- and isoproterenol-induced voltage-gated L-type calcium channel activation. These studies demonstrate that activation of CXCR4 results in a direct negative inotropic modulation of cardiac myocyte function. The specific mechanism of action involves alterations of calcium channel activity on the membrane. The presence of functional CXCR4 on cardiac myocytes introduces a new target for treating cardiac dysfunction.

Fig. 1

Activation of CXCR4 blunts PM response to Ca²⁺. Murine PM were subjected to two graded stepwise Ca²⁺ challenges separated by wash-out. PM exposed to diluent before the second Ca²⁺ challenge demonstrate similar responses when compared to the first challenge. (A) PM exposed to CXCL12 (125 ng/ml) demonstrated a blunted response during the second Ca²⁺ challenge (B).

Fig. 2

Activation of CXCR4 decreases CM contractility. Adult rat CM were exposed to CXCL12 (125 ng/ml) for either 5, 10 or 60 min prior to a stepwise Ca²⁺ challenge (A, B) or to increasing ISO concentrations after a 5-min exposure to CXCL12 (C). In both experiments, control CM were exposed to diluent only. The average percent increase in shortening (A) and average peak Ca²⁺ transients (B) during the Ca²⁺ challenge were significantly lower in CM exposed for either 5 or 10 min to CXCL12. No difference in shortening or Ca²⁺ transient was detected in CM exposed for 60 min to CXCL12 (A and B). Percent increase in fractional shortening in the CM is shown as a function of increasing [ISO] (0.001–10 μM) (C). CXCL12 exposed CM demonstrated a significant decrease in shortening response. ISO experiments were performed in triplicate.

Fig. 3

CXCR4 is present on adult rat CM. Alpha-actinin (red), CXCR4 chemokine receptor (green) and DAPI-stained nuclei (blue) were visualized in rodent and human myocytes. A single rat CM was stained one h after isolation with a polyclonal anti-rat CXCR4 Ab (green) and an anti-α-actinin Ab (red) and examined by fluorescent microscopy (top panel). Higher magnification (inset A) reveals a punctate distribution of CXCR4 on the surface (arrowheads). The CXCR4 Ab was omitted and only the fluoresceinated anti-rabbit-Cy2 Ab was applied (inset B). Normal human cardiac myocyte also demonstrate a similar pattern of CXCR4 chemokine receptor staining (bottom panel).

Fig. 4

Effects of CXCL12 on CM are mediated through CXCR4. Adult rat CM were pretreated with AMD3100 for 1 h and CXCL12 (125 ng/ml) for 10 min prior to a graded stepwise Ca²⁺ challenge. A representative tracing for fractional shortening (A) and peak Ca²⁺ transients during stimulation (B) are presented. Experiments were performed in triplicate.

Fig. 5

Adenoviral infection significantly increases expression of CXCR4 on rat CM. Adult rat CM were visualized to detect GFP fluorescence 24 (A) or 48 h (B) after infection with Ad.CXCR4. Co-expression of GFP demonstrates visually that CXCR4 gene is being expressed in the cells. A dose–response infection was performed in order to show significant increase in CXCR4 expression with increased MOI. The percentage of infected CM was determined as the number of GFP positives cells against the total cells counted in the bright-field. Different magnifications were used for optimal visualization of the cells. The bar graphs show that there was a significant increase in CXCR4-infected CM even at the lowest MOI.

Fig. 6

Overexpression of CXCR4 decreases CM contractility. Both CM infected with Ad.CXCR4 and control uninfected CM were exposed to CXCL12 (125 ng/ml) and then subjected to a graded stepwise Ca²⁺ challenge. The average percent shortening (A) at 37 °C at 1 Hz and average peak Ca²⁺ transients (B) at increasing external Ca²⁺ are presented. *p<0.05 vs. uninfected control in the presence of CXCL12.

Fig. 7

CXCL12 modulates forskolin (FSK)-induced Ca²⁺ channel activation in CM. Whole-cell recording of inward Ca²⁺ currents from isolated rat ventricular CM with 1 mM CaCl₂ in the bath solution. L-type Ca²⁺ currents were activated with the step voltage clamp protocol shown in the top panel. Currents were mostly blocked by Cd (0.1 mM, lower panel in red) (A). I-V plot of the L-type Ca²⁺ current in control conditions (blue) and in 0.1 mM Cd (red) (B). Time course of Ca²⁺ current in response to the application of FSK (1 μm) and CXCL12 (100 ng/ml). The top panel illustrates the voltage protocol and typical current traces under each condition. The inward Ca²⁺ currents were activated by a repeating voltage step from −50 mV to 0 mV. Inward current values were measured at the inward peak. The bottom panel illustrates the relative changes of peak currents during 3-min applications of FSK (1 μM) indicated by the red horizontal lines. CM were exposed for approximately 5 min to either CXCL12 (FSK-CXCL12-FSK) or diluent (FSK-CON-FSK), as indicated by the blue horizontal line, before the second FSK application (C). Summary of the CXCL12 attenuation of the FSK-induced Ca²⁺ current stimulations is shown (n=5) (D).

Fig. 8

CXCL12 modulates β-adrenergic Ca²⁺ channel activation in CM. Whole-cell inward Ca²⁺ currents were recorded from isolated adult rat ventricular CM with 1 mM Ca²⁺ in the bath solutions. Ca²⁺ currents were activated with a protocol ramping from −50 mV to +50 mV from a holding potential of −60 mV. I-V plots of Ca²⁺ currents against membrane potentials recorded in control condition (blue), ISO (1 μM, red) and in CdCl2 (0.1 mM, black) (A). Representative time course record of the Ca²⁺ current from CM treated with ISO alone (1 μM, top panel), or pre-incubated with CXCL12 (100 ng/ml) for 5 min prior to ISO treatment (lower panel). Applications of ISO, (1 μM), CXCL12 (100 ng/ml) and CdCl₂ (0.1 mM) are indicated by the bars. Data values for analysis were taken from the point before ISO and 200 s after ISO application, as indicated by the arrows in each panel B. Summary of the effects of CXCL12 on ISO-induced Ca²⁺ current increases (n=11) (C).