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J Appl Physiol 101: 778-784, 2006. First published June 8, 2006; doi:10.1152/japplphysiol.01631.2005
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Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A-dependent pathway in murine ventricular myocytes

Feng Dong,1 Meghan M. Taylor,2 Willis K. Samson,2 and Jun Ren1

1Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming; and 2Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri

Submitted 28 December 2005 ; accepted in final form 25 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intermedin (IMD), also called adrenomedullin-2, is a 47-amino acid peptide from the calcitonin gene-related peptide (CGRP)/adrenomedullin family of peptides. Recent studies suggest that IMD may participate in the regulation of cardiovascular function and fluid and electrolyte homeostasis. To evaluate the role of IMD on cardiomyocyte contractile function, electrically paced murine ventricular myocytes were acutely exposed to IMD, and the following indexes were determined: peak shortening (PS), time to PS, time-to-90% relengthening, and maximal velocity of shortening and relengthening. Intracellular Ca2+ was assessed using fura 2-AM fluorescent microscopy. Our results revealed that IMD (10 pM to 10 nM) significantly increased PS and maximal velocity of shortening and relengthening in ventricular myocytes, the maximal effect of which (~46%) was somewhat comparable to those elicited by CGRP (1 nM) and adrenomedullin (100 nM). Exposure of IMD significantly shortened time-to-90% relengthening without affecting time to PS, similar to CGRP and adrenomedullin. IMD also enhanced intracellular Ca2+ release, with a maximal increase of ~50%, and facilitated the intracellular Ca2+ decay rate. The IMD-induced effects were abolished by the protein kinase C inhibitor chelerythrine (1 µM), downregulation of protein kinase C using phorbol 12-myristate 13-acetate (1 µM), and the protein kinase A inhibitor H89 (1 µM). Our data suggest that IMD acutely augments cardiomyocyte contractile function through, at least in part, a protein kinase C- and protein kinase A-dependent mechanism.

cardiac myocyte; shortening; relengthening

ADRENOMEDULLIN (AM) and calcitonin gene-related peptide (CGRP) are homologous peptides with important roles in fluid balance, electrolyte homeostasis, and cardiovascular, neuronal, and endocrine function (2, 5, 20). Due to their sequence analogy, the calcitonin receptor-like receptor (CRLR) may serve either as a CGRP receptor or as an AM receptor, depending on the coexpression of one of three receptor activity-modifying proteins (RAMP1–3). It is believed that the CRLR/RAMP1 complex functions as a specific CGRP receptor, whereas AM preferentially binds to CRLR/RAMP2 or CRLR/RAMP3 (8, 10). Both CGRP and AM play key roles in the regulation of cardiac function, including a positive inotropic response on the heart (1, 7, 9, 16). Recently, a novel member of the calcitonin/CGRP/AM family of peptides, namely intermedin (IMD; or AM-2), was identified (14, 17). The human IMD gene is believed to encode a prepropeptide of 148 amino acids, which may generate a 47-amino acid mature peptide (IMD1–47) and a shorter 40-amino acid one (IMD8–47) by proteolytic cleavage at the NH2-terminal proximate basic residues. Intravenous infusion of IMD, like AM and CGRP, has been reported to reduce blood pressure and increase heart rate, suggesting that the peptide may be an important regulator for cardiac function (14, 17, 18). Unlike CGRP and AM, which exhibit a preferential stimulation of CRLR when coexpressed with RAMP1 and RAMP2 or RAMP3, respectively, IMD represents a nonselective agonist for the RAMP coreceptors (14). IMD and the CRLR/RAMP complexes are expressed in myocardium (14, 17, 18, 26), indicating the peptide may act in an autocrine or paracrine manner to regulate heart function. Nonetheless, the role of IMD on cardiac contractile function has only recently been explored (12, 24, 25). The aim of this study was to determine the direct effects of IMD on cardiomyocyte contractile function and potential signaling mechanisms involved by using isolated murine ventricular myocytes.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of murine ventricular myocytes.   The experimental procedure used in this study was approved by the University of Wyoming Animal Use and Care Committee (Laramie, WY). Briefly, adult male FVB mice (body weight ~22 g, a total of 7 mice were used for this study) were anesthetized with ketamine/xylazine (5:3, 1.32 mg/kg ip). Hearts were rapidly removed and perfused (at 37°C) with Krebs-Henseleit bicarbonate buffer (in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 HEPES, 11.1 glucose, pH 7.4). The heart was then perfused for 20 min with Krebs-Henseleit bicarbonate containing Liberase Blendzyme 4 (Hoffmann-La Roche, Indianapolis, IN). After perfusion, the left ventricle was removed and minced. The cells were filtered through a nylon mesh (300 µm). Extracellular Ca2+ was added incrementally back to 1.25 mM. Isolated myocytes were maintained in a serum-free medium before experimentation (3).

Cell shortening/relengthening.   Mechanical properties of ventricular myocytes were assessed using an IonOptix MyoCam soft-edge system (IonOptix, Milton, MA) (3). In brief, cells were superfused with a buffer containing (in mM) 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, at pH 7.4. The cells were field stimulated at 0.5 Hz. The myocyte was displayed on a computer monitor using an IonOptix MyoCam camera, which rapidly scans the image area every 8.3 ms such that the amplitude and velocity of shortening/relengthening were recorded with good fidelity. Cell shortening and relengthening were assessed using the following indexes: peak shortening (PS) amplitude, time to 90% PS (TPS), time to 90% relengthening (TR90), and maximal velocities of shortening (+dL/dt) and relengthening (–dL/dt).

Intracellular fluorescence measurement.   Myocytes were loaded with fura 2-AM (0.5 µM) for 10 min, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (Ionoptix) (3). Myocytes were imaged through an Olympus (model IX-70) fluor x40 oil objective. Cells were exposed to light emitted by a 75-W lamp and passed through either a 360- or a 380-nm filter (bandwidths were ±15 nm), while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm after first illuminating cells at 360 nm for 0.5 s then at 380 nm for the duration of the transient period (1,000-Hz sampling rate). The 360-nm excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular Ca2+ concentration were inferred from the ratio of the fura 2 fluorescence intensity (FFI) at two wavelengths.

Experimental protocols.   Myocytes (either fura 2-AM loaded or nonloaded) were equilibrated for a minimum of 1 h before IMD introduction. IMD (IMD1–47, rat, Phoenix Pharmaceuticals, Belmont, CA) was added to the contractile buffer at the concentration tested for 20 min before myocyte contractile function was determined in electrically paced (0.5 Hz) cells. For comparison purpose, cohorts of cardiomyocytes were also exposed to CGRP (1 nM) or AM (100 nM) for 20 min to evaluate the effect of these peptides on myocyte contractile function. To elucidate whether protein kinase C (PKC) or protein kinase A (PKA) plays a role in IMD-induced cardiac contractile response, cells in some experiments were pretreated with the PKC inhibitor chelerythrine chloride (1 µM) or the PKA inhibitor H89 (1 µM) for 20 min before IMD (10 nM) was applied for another 20 min. Inhibition of PKC was also achieved via PKC downregulation by incubating cells with phorbol 12-myristate 13-acetate (PMA; 1 µM) for 60 min (13) before IMD (10 nM) application for 20 min.

Statistical analyses.   For each experimental series, data were presented as means ± SE. Statistical significance (P < 0.05) for each variable was determined by repeated-measures ANOVA for concentration-dependent response or effect of kinase inhibitors on the IMD response. A Tukey's post hoc test was used where appropriate.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of IMD on myocyte PS.   The average resting cell length of cardiomyocytes used in this study was 115.6 ± 0.8 µm (n = 594 cells). IMD exposure did not affect cell shape and percentage of variable cells (~70%) over the concentration range tested (data not shown). Representative traces depicting the effect of IMD (10 nM) and its analogs CGRP (1 nM) and AM (100 nM) on myocyte shortening following 20 min of exposure are shown in Fig. 1A. All three peptides enhanced myocyte shortening amplitude in a comparable manner. None of these peptides affected the resting cell length (Fig. 1B). IMD, CGRP, and AM significantly enhanced PS amplitude, +dL/dt, and –dL/dt, with maximal increases between 25.4 and 46.4% (Fig. 1, C and D). The threshold of IMD-induced augmentation in PS and ±dL/dt was between 50 and 100 pM followed by a plateau (~46% increase) between 100 pM and 10 nM (Fig. 1, C and D). IMD significantly decreased TR90 without affecting TPS, similar to those elicited by CGRP and AM (Fig. 1, E and F). Consistent with its response on PS and ±dL/dt, the threshold of IMD-induced shortening of TR90 was between 50 and 100 pM (Fig. 1D). Interestingly, representative traces in Fig. 2A depict that IMD (10 nM)-induced positive effect on cell shortening was nullified by the PKC inhibitor chelerythrine (1 µM) or the PKA inhibitor H89 (1 µM). Although resting cell length was not affected by IMD, the kinase inhibitors or PKC activator PMA (which downregulates PKC) (Fig. 2B), the IMD-elicited effects on PS, ±dL/dt, and TR90 were abolished by chelerythrine and H89, indicating involvement of both kinase pathways in IMD-exerted response. Neither chelerythrine nor H89 itself elicited any significant effect on cardiac contractile response (Fig. 2). To confirm the involvement of PKC pathway, myocytes were incubated with PMA (1 µM) for 60 min to downregulate PKC kinase (13). Although PMA treatment itself significantly attenuated PS and ±dL/dt as well as shortened TPS (but not TR90) duration, IMD failed to elicit any positive contractile response following PMA treatment (compared with the PMA group) (Fig. 2, C–F). In addition, the IMD-induced shortening of TR90 was ablated by PMA treatment (Fig. 2F). These data confirmed that PKC is likely involved in IMD-induced cardiac contractile response.


Figure 1
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  		Fig. 1. Effects of intermedin (IMD; 10 pM to 10 nM), calcitonin gene-related peptide (CGRP; 1 nM), and adrenomedullin (AM; 100 nM) on cardiomyocyte contractile function in isolated murine ventricular myocytes. A: representative traces depicting effects of IMD (10 nM), CGRP (1 nM), and AM (100 nM) on myocyte shortening. B: resting cell length. C: peak shortening amplitude (normalized to resting cell length). D: maximal velocity of shortening and relengthening (±dL/dt). E: time to peak shortening (TPS). F: time to 90% relengthening (TR90). Values are means ± SE; n = 33–42 cells per group. *P < 0.05 vs. control.

 

Figure 2
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  		Fig. 2. Effect of the PKC inhibitor chelerythrine (1 µM) and downregulation of PKC with 60-min pretreatment of phorbol 12-myristate 13-acetate (PMA, 1 µM) and the PKA inhibitor H89 (1 µM) on IMD (10 nM)-induced mechanical response in isolated murine ventricular myocytes. A: representative traces depicting the effects of the PKC inhibitor (PKCI) and H89 on IMD-elicited cell-shortening response. B: resting cell length. C: peak shortening amplitude (normalized to resting cell length). D: ±dL/dt. E: TPS. F: TR90. Values are means ± SE; n = 33–41 cells per group. *P < 0.05 vs. control. #P < 0.05 vs. 10 nM IMD group.

 
Effect of IMD on intracellular Ca2+ properties.   The Ca2+ fluorescence indicator fura 2-AM was utilized to evaluate the effect of IMD on intracellular Ca2+ handling. Resting and peak intracellular Ca2+ levels were assessed in electrically stimulated cells (at 0.5 Hz). Representative traces shown in Fig. 3A illustrate a positive effect of IMD (10 nM) on intracellular Ca2+ transients. Increasing concentrations of IMD (0–10 nM) did not affect the resting levels of intracellular Ca2+, determined by baseline FFI (Fig. 3B). Similar to its effect on PS, change in FFI ({Delta}FFI) in response to the electrical stimuli was enhanced by IMD (Fig. 3C). However, the threshold of IMD-elicited positive response on {Delta}FFI was between 1 and 10 nM, reflecting a rightward shift from its threshold on cell shortening response. Intracellular Ca2+ clearance velocity (evaluated by intracellular Ca2+ transient decay rate) was facilitated by IMD, with a threshold between 50 and 100 pM (Fig. 3D). Similar to the mechanical properties, IMD-induced effects on {Delta}FFI and intracellular Ca2+ transient decay rate were abolished by the PKC inhibitor chelerythrine. Chelerythrine itself did not elicit any significant effect on intracellular Ca2+ transients.


Figure 3
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  		Fig. 3. Effect of IMD (10 pM to 10 nM) on intracellular Ca2+ transients in isolated murine ventricular myocytes. A: representative traces from control, 10 nM IMD-treated, and 10 nM IMD + chelerythrine (1 µM)-treated cells. B: resting intracellular Ca2+ concentration ([Ca2+]i) represented by resting fura 2 fluorescence intensity (FFI). C: change in FFI from baseline ({Delta}FFI). D: intracellular Ca2+ transient decay rate. Values are means ± SE; n = 21–24 cells per group. *P < 0.05 vs. control. #P < 0.05 vs. 10 nM IMD group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Initial work from our laboratory (18) and other groups (17) has demonstrated the presence of IMD mRNA and peptide immunoreactivity in the heart. Our present study demonstrates that IMD augments cardiac contractile function and electrically stimulated intracellular Ca2+ transients in isolated murine ventricular myocytes. This raises the possibility that IMD produced in the heart acts in vivo via an autocrine or paracrine mechanism to augment cardiac function, in particular cardiac contractility. This notion is further supported by the evidence that the IMD receptor, the CRLR/RAMP complexes, are expressed in the myocardium (22). Indeed, Yang and colleagues (24) have demonstrated that IMD protects isolated rat hearts from ischemia/reperfusion injury. In their Langendorff preparations, reperfusion with IMD significantly attenuated the detrimental effects of global ischemia on left ventricular function (24). Cardiac effects of IMD were also reported by Pan and colleagues in intact animals following intravenous administration and in isolated heart preparations (12). However, their study suggested an inhibitory effect of IMD in contrary to the positive effect seen in this study and elsewhere (24, 25). Pan and colleagues proposed that the myocardial action of IMD was mediated via G protein coupling to adenylate cyclase, since levels of cAMP in the tissues and perfusion buffers were increased in the presence of the peptide. This is somewhat surprising since activation of adenylate cyclase would be expected to be trigger positive, not negative, chronotropic/inotropic effects. Earlier study from our laboratory demonstrated that the beta-adrenergic agonist isoproterenol (1 µM) elicits an ~50% increase in PS amplitude in the same murine cardiomyocytes (11), similar to that elicited by IMD. In our hands, the IMD-induced enhancement of contractile function was ablated by inhibition of either PKA or PKC, suggesting that a PKA-/PKC-dependent mechanism is likely involved in IMD-elicited cardiac contractile response.

Our study revealed that IMD elicits positive contractile response in isolated murine cardiomyocytes in a manner similar to that elicited by CGRP and AM. In fact, IMD has been reported to activate CGRP and AM receptors (CRLR in association with RAMP1, or RAMPs 2 or 3, respectively) (14, 17), and both CGRP and AM have been demonstrated to exert positive inotropic effects in heart (1, 9, 16), potentially via the CRLR/RAMP complexes (10). However, the vasodilatory effect of intravenous IMD can only be partially attenuated by CGRP or AM antagonists (14, 18), which seems to be in line with our recent observation that IMD may bind and activate a unique receptor (19). Furthermore, although IMD may be associated with cAMP accumulation in the hearts (12), IMD may actually decrease cAMP accumulation in the absence or presence of phosphodiesterase inhibition in at least one tissue other than the heart (19). Thus tissue specificity and second messenger heterogeneity may exist depending on cell type studied, particularly when isolated cell types are examined as opposed to whole organs or the intact organism. Therefore, it is possible that IMD acts through non-CRLR/RAMP receptor complexes to produce its procontractile effects in the myocardium and that, unlike the AM and CGRP, the action of IMD is mediated via Ca2+ mobilization by the inositol trisphosphate generated once PLC is activated. This speculation of PLC involvement in IMD-induced cardiomyocyte contractile response is supported by our observation that chelerythrine and PKC downregulation using PMA nullified IMD-elicited contractile response (intracellular Ca2+ transients for chelerythrine as well). PKC activation is known to stimulate cardiomyocyte contractile function via improved intracellular Ca2+ handling and increased myofilament protein phosphorylation (6). Nevertheless, our data showed that prolonged PKC activation by PMA itself depressed cardiac contractile function (reduced PS, ±dL/dt, and shortened TPS). It has been speculated that inhibition of PKC activity in the hearts may improve systolic and diastolic function, whereas chronic activation of PKC may cause a lethal restrictive cardiomyopathy with marked interstitial fibrosis (4). Although direct evidence for IMD-induced activation of PKC in hearts is still lacking, it may be speculated that excessively high levels of IMD may cause chronic PKC activation and promote the development of heart failure. Clearly, this hypothesis on PKC activation in response to IMD merits further research in the setting of both physiology and pathophysiology.

In this study, acute IMD exposure to murine ventricular myocytes directly enhanced PS by ~45%. The IMD-induced maximal increase in myocyte shortening amplitude was associated with an augmentation of ±dL/dt as well as intracellular Ca2+ release ({Delta}FFI) in response to electrical stimuli. Although the mechanism(s) for IMD-induced augmentation of cardiomyocyte contractile function is not clear, the effect of IMD on PKC, PKA, as well as intracellular Ca2+ release may all play a crucial role. Results from our present study demonstrated that IMD significantly enhanced {Delta}FFI with a threshold between 1 and 10 nM, which reflects a rightward shift from its threshold on cell-shortening response (between 50 and 100 pM). This discrepancy in the threshold of IMD-elicited mechanical and intracellular Ca2+ responses suggests that certain intracellular Ca2+-independent mechanism may be involved in IMD-induced cardiac contractile response (such as myofilament Ca2+ response or actin-myosin cross-bridge linking). It is possible that IMD-shortened TR90 may be related to faster intracellular Ca2+ clearance (indicated by a smaller decay rate), although evidence on the effect of IMD on cytosolic Ca2+ extrusion and cytosolic Ca2+ extrusion machineries [e.g., Na+/Ca2+ exchanger and sarcoplasmic reticulum-Ca2+-ATPase (SERCA)] is still lacking. Another scenario that can be postulated for IMD-induced shortening of TR90 is the enhanced PKA- or PKC-dependent phosphorylation of phospholamban (which unblocks SERCA to facilitate Ca2+ clearing as seen in our intracellular Ca2+ transients) (15, 23). The IMD-induced increase of {Delta}FFI may be a result of facilitated Ca2+ release from intracellular Ca2+-storage organelles, such as the sarcoplasmic reticulum and mitochondria. These findings are consistent with the ability of AM and CGRP to rapidly facilitate intracellular Ca2+ release and enhance cardiac contractility via mechanisms involving Ca2+ release from intracellular ryanodine- and thapsigargin-sensitive Ca2+ stores, activation of PKA and PKC, as well as Ca2+ influx through L-type Ca2+ channels (7, 16).

In conclusion, our study reveals that IMD stimulates cardiomyocyte contractile function mediated, at least in part, by increased intracellular Ca2+ transients and activation of PKC as well as PKA. Further investigation is warranted to examine the effect of IMD on sarcoplasmic reticulum Ca2+ release, membrane Ca2+ channels, and Ca2+ regulatory proteins, which should be essential to the understanding of the role of IMD on cardiac contractile function. The physiological relevance of the IMD-elicited cardiac stimulatory effect is unknown at this time. However, our data match findings in isolated heart preparations (25), suggesting that, like CGRP and AM, IMD may be a physiologically important, cardioprotective peptide. The procontractile actions of IMD may also complement additional actions previously demonstrated in brain to augment sympathetic activity (18), stimulate stress hormone secretion (21), and, in so doing, play an integrative role in the response to immediate danger or perceived stress.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Salary support for Feng Dong was from the American Heart Association Pacific Mountain Affiliate (0355521Z to J. Ren).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Ren, Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, Univ. of Wyoming, 1000 E. Univ. Ave., Dept. 3375, Laramie, WY 82071-3375 (e-mail: jren{at}uwyo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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