Differential regulation of inotropy and lusitropy in overexpressed Gsα myocytes through cAMP and Ca²⁺ channel pathways

We investigated the mechanisms responsible for altered contractile and relaxation function in overexpressed Gsα myocytes. Although baseline contractile function (percent contraction) in Gsα mice was similar to that of wild-type (WT) mice, left ventricular myocyte contraction, fura-2 Ca²⁺transients, and Ca²⁺ channel currents (I_Ca) were greater in Gsα mice in response to 10^–8 M isoproterenol (ISO) compared with WT mice. The late phase of relaxation of the isolated myocytes and fura-2 Ca²⁺ transients was accelerated at baseline in Gsα but did not increase further with ISO. In vivo measurements using echocardiography also demonstrated enhanced relaxation at baseline in Gsα mice. Forskolin and CaCl₂ increased contraction similarly in WT and Gsα mice. Rp-cAMP, an inhibitor of protein kinase, blocked the increases in contractile response and Ca²⁺ currents to ISO in WT and to forskolin in both WT and Gsα. It also blocked the accelerated relaxation in Gsα at baseline but not the contractile response to ISO in Gsα myocytes. Baseline measurements of cAMP and phospholambation phosphorylation were enhanced in Gsα compared with WT. These data indicate that overexpression of Gsα accelerates relaxation at end diastolic but does not affect baseline systolic function in isolated myocytes. However, the enhanced responses to sympathetic stimulation partly reflect increased Ca²⁺ channel activity; i.e the cellular mechanisms mediating these effects appear to involve a cAMP-independent as well as a cAMP-dependent pathway.

The sympathetic nervous system plays a major role in the regulation of cardiovascular function under various stress conditions, such as hypotension, exercise, and the fight-or-flight reaction, by releasing catecholamines, which, in turn, activate the β-adrenergic receptor–Gs–adenylyl cyclase (βAR-Gs-AC) pathway. To understand the physiological and pathological mechanisms of this cascade when the stimulation is chronic, murine models have been created by altering expression of components of the βAR signaling pathway (1–9). These models all demonstrated enhanced efficacy of the βAR-Gs-AC signaling pathway (1–9). However, the chronic augmentation of βAR signaling in the model with overexpression of cardiac Gsα leads to myocyte hypertrophy and cardiomyopathy as these animals age (2, 4, 5), which differs from what has been reported in the other models (8, 9). Another controversial issue is whether Gsα can alter L-type Ca²⁺ channel activity in a cAMP-PKA–independent manner. Prior studies exist supporting such a mechanism in vitro (10–13), although there is another that contradicts this position (14). The current model of overexpressed Gsα presents a unique opportunity to reconcile this controversy. To address these mechanistic questions, it is necessary to study the contractile function of isolated myocytes, which has not been done previously in the model of overexpressed Gsα (1–7). Furthermore, all prior studies have concentrated on inotropic function in vivo (2–5), neglecting another important controlling mechanism, lusitropic function. The isolated myocyte technique lends itself to examination of that aspect of myocardial function as well. Finally, it was important to conduct the present investigation in adult as opposed to neonatal (7) myocytes, because (a) it is possible to examine contractile and relaxation function as well as Ca²⁺ channel activity in adult cells, and (b) it is well known that βAR-Gs-AC regulation differs markedly in neonatal animals (15–17).

The first goal of this study was to investigate the regulation of isolated left ventricular (LV) myocyte contractility by the βAR-Gs-AC pathway and the extent to which myocyte contractile function is altered by overexpression of cardiac Gsα, as this approach will provide an assessment of intrinsic contractile function independent of the extracellular matrix and hemodynamic and neurohormonal effects. As part of this goal, it was important to determine whether lusitropic function was affected in a fashion parallel to inotropic function in the isolated myocytes. Once it was determined that lusitropic function was altered at baseline both in isolated myocytes and in vivo, the mechanism was investigated by measuring baseline cAMP and phospholamban phosphorylation. The next goal of this study was to determine whether the mechanism for the enhanced effects in response to βAR stimulation involved an action on the Ca²⁺ channel, potentially independent of cAMP. This was accomplished using a dual approach. First, Ca²⁺ channel function was assessed directly using patch-clamp techniques. Second, the effects of βAR stimulation were examined after the cAMP pathway was blocked with Rp-cAMP, an inhibitor of protein kinase A (PKA), which should abolish the enhanced inotropic effects induced by βAR stimulation of AC via Gs.

Baseline contractile and relaxation function. Figure 1 shows representative contraction/relaxation and Ca²⁺ transient recordings at baseline in WT and Gsα. Although peak amplitude of contraction and Ca²⁺ transients were not different from WT myocytes, the late relaxation phase in Gsα myocytes was significantly shorter; this was associated with accelerated Ca²⁺ uptake. As summarized in Table 1, indices of systolic function (percent contraction and –dL/dt) were similar in Gsα and WT, as was one index of diastolic function, the rate of relaxation (+dL/dt). However, as noted in Figure 1, the late phase of relaxation was accelerated in Gsα: the time for 70% recovery of relaxation was less (P < 0.05) in Gsα (46 ± 7 ms) compared with WT (57 ± 7 ms) (Table 1). Similar data were observed for the Ca²⁺ transients. The amplitude of systolic Ca²⁺ uptake function was similar at baseline for WT myocytes (0.51 ± 0.06) and Gsα myocytes (0.55 ± 0.09), whereas the late recovery was significantly faster in Gsα-overexpressed myocytes. For example, the time to 70% recovery of the Ca²⁺ transient was less (P < 0.05) in Gsα myocytes (94 ± 9 ms) compared with myocytes from WT mice (139 ± 17 ms).

Figure 1

Superimposed representative length (a) and Ca²⁺ transient (b) recordings from a WT myocyte (thin line) and Gsα-overexpressed myocyte (thick line). Neither myocyte contractile function nor systolic fura-2 signaling was affected in overexpressed Gsα myocytes. However, the recovery in late diastole was faster in Gsα, as noted by the time to 70% recovery of relaxation (circles on tracings). WT, wild-type.

Table 1

Contractile and relaxation function in response to ISO (10^–8 M) in myocytes from WT and Gsα mice

To determine whether results of contractile and relaxation function obtained in isolated myocytes are similar to what is observed in vivo, systolic and diastolic function was also assessed by echocardiography. Consistent with the in vitro data, we found a significant (P < 0.05) increase in relaxation, using the E/A ratio as an index of diastolic function (Gsα: 1.29 ± 0.04; WT: 1.19 ± 0.02). However, ejection fraction as an index of LV systolic function in vivo was not different at baseline in the Gsα and WT mice (Gsα: 71 ± 2%, n = 3; WT: 73 ± 2%, n = 3).

Contraction and relaxation responses to isoproterenol, forskolin, and CaCl₂. Figure 2 shows representative contraction/relaxation and Ca²⁺ transient recordings in response to isoproterenol (ISO) in Gsα and WT mice. Peak systolic contraction and fura-2 signaling were enhanced in Gsα after ISO. The differences in late diastolic recovery between Gsα and WT mice were no longer apparent, because this part of relaxation was accelerated by ISO in WT but not in Gsα mice. Figure 3 compares dose–response data for contractile function (percent contraction, –dL/dt_max) and relaxation function (+dL/dt_max) in Gsα and WT, whereas Table 1 compares the data at one dose. In response to ISO, myocyte contractile indices and rate of relaxation (+dL/dt_max) were increased more (P < 0.05) in Gsα than in WT myocytes, and the dose–response curve to ISO was shifted (Figure 3). The slopes of the dose responses for both contractile and relaxation indexes were significantly greater in Gsα myocytes (P < 0.05) compared with WT myocytes. For example, at ISO 10^–8 M, percent contraction in Gsα myocytes was increased (P < 0.05) (14.2 ± 0.9 vs. 8.4 ± 1.1%) and the maximum rate of contraction (–dL/dt_max) was increased (P < 0.05) (–885 ± 114 vs. –532 ± 84 μm/s), compared with myocytes from WT. The maximum rate of relaxation (+dL/dt_max) in Gsα myocytes was also increased (P < 0.05) compared with WT myocytes (808 ± 90 vs. 465 ± 55 μm/s) (Table 1). Of note, the enhanced contractile responses to ISO were associated with increased Ca²⁺ transients measured by fura-2 (Figure 2). The amplitude of the Ca²⁺ signal in Gsα myocytes was significantly increased (from 0.55 ± 0.09 to 0.87 ± 0.08; P < 0.05) compared with WT controls (from 0.51 ± 0.06 to 0.67 ± 0.04; P < 0.05). ISO reduced the time to 70% recovery of relaxation in WT (from 57 ± 7 to 43 ± 2 ms) but did not further reduce the time to 70% recovery of relaxation in Gsα (from 46 ± 7 to 47 ± 6 ms). Similarly, the time for 70% decay of the Ca²⁺ transient was reduced (P < 0.05) with ISO in WT myocytes (from 139 ± 17 to 112 ± 11 ms) but not in Gsα myocytes (from 94 ± 9 to 82 ± 5 ms.). To determine whether the enhanced ISO responses observed in Gsα myocytes were a consequence of an action proximal to AC activation, the effects of forskolin on contractile function were measured. Forskolin elicited similar increases in contractile and relaxation function in Gsα and WT mice (Figure 4). Furthermore, non–βAR-mediated inotropic stimulation by CaCl₂ was not altered in Gsα myocytes (Figure 5).

Figure 2

Superimposed representative contraction (a) and Ca²⁺ transient (b) recordings in response to ISO (10^–8 M) in WT (thin line) and Gsα (thick line). In response to ISO, the enhanced contractile function was associated with an increased Ca²⁺ transient in Gsα. Note that differences in late diastolic recovery were less apparent after ISO (compare with Figure 1), as the time to 70% recovery of relaxation was no longer accelerated in Gsα. ISO, isoproterenol.

Figure 3

Enhanced contractile (percent contraction, –dL/dt) and relaxation (+dL/dt) function in response to ISO in Gsα compared with WT, and the upward shift of the dose–response curves. *P < 0.05 vs. respective WT.

Figure 4

Contraction and relaxation in response to forskolin (10^–8 to 10^–6 M). Forskolin elicited similar increases in contractile and relaxation function in Gsα and WT.

Figure 5

Contraction and relaxation in response to CaCl₂ (2 and 3 mM). CaCl₂ elicited similar increases in contractile and relaxation function in Gsα and WT.

Contractile and relaxation function in the presence of Rp-cAMP. As shown in Table 2 and Figure 6, Rp-cAMP completely blocked LV contractile and relaxation function in response to ISO in WT myocytes but not in Gsα myocytes. Interestingly, after Rp-cAMP, the differences in the time to 70% recovery of the relaxation between WT and Gsα myocytes at baseline were abolished (WT: 62 ± 14 ms ; Gsα: 57 ± 4 ms) because Rp-cAMP prolonged the time for 70% recovery in Gsα myocytes (from 47 ± 3 to 57 ± 4 ms) (P < 0.005). However, increases in contractile function in both Gsα and WT myocytes in response to forskolin (10^–7 M) were completely abolished in the presence of Rp-cAMP (Figure 6). Thus, the increased contractile function in Gsα myocytes in response to ISO is not simply a result of enhanced AC activity but, rather, may involve a cAMP-independent mechanism. We examined the L-type Ca²⁺ channel to determine whether its regulation was similarly altered in Gsα myocytes.

Figure 6

Contractile function in response to ISO (a) and forskolin (b) in the presence of Rp-cAMP. Rp-cAMP blocked the increase in percent contraction in response to ISO in WT and to forskolin in both WT and Gsα, but not the response to ISO in Gsα myocytes. *P < 0.05 vs. respective baseline.

Table 2

Contractile and relaxation function in response to ISO (10^–8 M) following Rp-cAMP (200 μm) in myocytes from WT and Gsα mice

Ca²⁺ channel function in response to ISO and the effects of Rp-cAMP. In an attempt to characterize more fully the mechanisms for the enhanced inotropy in Gsα myocytes, Ca²⁺ channel activity was measured in myocytes from Gsα (n = 5) and WT (n = 5) mice. Although the current–voltage (I–V) relationships in Gsα myocytes are similar to those in WT myocytes, the Ca²⁺ channel current (I_Ca) density was significantly less (P < 0.01) (Figure 7). However, the response to dihydropyridines was not altered. For example, 0.1 μM Bay K 8644 (a dihydropyridine agonist) increased I_Ca and also shifted the I–V relationship to negative potentials (Gsα: 2.1 ± 0.3–fold and 12.3 ± 1.4 mV, n = 8; WT: 2.3 ± 0.1–fold and 14.4 ± 0.7 mV, n = 17). Similarly, a dihydropyridine antagonist, nifedipine (1 μM), reduced I_Ca amplitude to 9.59 ± 0.03% of baseline in Gsα (n = 5), similar to that observed in WT (11.20 ± 0.02% of baseline, n = 5). Interestingly, the maximal I_Ca response to ISO in Gsα was significantly higher compared with WT myocytes (Gsα: 3.1 ± 0.2–fold, n = 16; WT: 2.3 ± 0.1–fold, n = 42; P < 0.001) (Figure 8). However, there was no difference in the effects of forskolin (5 μM) (Gsα: 2.0 ± 0.1–fold, n = 14; WT: 2.1 ± 0.1–fold, n = 6), indicating that the enhanced responsiveness reflects signaling at the level of Gsα. To support this hypothesis, the effects of ISO were reexamined in the presence of Rp-cAMP (100 μM). Consistent with the enhanced response to ISO as measured by myocyte contraction, I_Ca response to ISO in the presence of Rp-cAMP was significantly higher in Gsα (P < 0.001) compared with WT myocytes (Figure 9).

Figure 7

Representative I_Ca recordings in WT (a) and Gsα (b) mice. Currents were elicited from a holding potential of –50mV to the indicated test potentials. (c) A comparison of the current–voltage relationships in WT and Gsa mice. I_Ca was normalized to the cell capacitance to give current densities (pA/pF). Average peak I_Ca densities for WT and Gsα mice were 8.4 ± 0.5 (pA/pF) and 6.6 ± 0.3 (pA/pF), respectively. Numbers correspond to number of cells. I_Ca, Ca²⁺ channel currents.

Figure 8

Concentration-dependent effects of ISO on I_Ca in WT and Gsα mice. The increase of current amplitude relative to baseline was plotted against ISO concentration. The increase in I_Ca amplitude in Gsα myocytes in response to ISO was significantly higher than in WT myocytes (P < 0.005). Data are mean ± SE from 16–42 cells.

Figure 9

Change to I_Ca response in the presence of Rp-cAMP. (a) Current traces were recorded in WT and Gsα myocytes from a holding potential of –50 mV to 0 mV and were superimposed before (open circles) and after ISO (filled circles). (b) Mean increase of I_Ca elicited by ISO assessed in the presence of Rp-cAMP, ISO, increased I_Ca in Gsα myocytes, but not in WT. Numbers correspond to number of cells. *P < 0.001 vs. respective WT.

Determination of cAMP concentration and Western blot analysis. The concentration of cAMP in the heart was significantly higher (P < 0.05) in Gsα mice (1.21 ± 0.08 pmol/mg tissue, n = 7) compared with WT mice (0.94 ± 0.08 pmol/mg tissue, n = 8). Furthermore, phospholamban phosphorylation (Ser¹⁶) was higher at baseline in myocytes from Gsα mice (Figure 10).

Figure 10

(a) Western blots of baseline phospholamban phosphorylation at Ser¹⁶ in isolated cardiac myocytes from WT and Gsα mice. (b) Baseline phospholamban phosphorylation at Ser¹⁶ in Gsα myocytes (n = 3) is increased compared with WT (n = 3). Y axis is in arbitrary units.

Activation of the sympathetic nervous system plays a major role in maintaining cardiovascular homeostasis by increasing inotropy, chronotropy, and lusitropy. These changes are mediated by activation of the βAR signaling pathway, leading to PKA activation and phosphorylation of intracellular proteins. A key target of PKA is the sarcolemmal L-type Ca²⁺ channel, which, when phosphorylated, enhances Ca²⁺ entry into the cell (30). Although this signaling pathway is clearly important in the acute and subacute maintenance of cardiovascular homeostasis under conditions of stress, the extent to which chronic stimulation of this pathway is beneficial or deleterious remains controversial (2). To understand the physiological and pathological mechanisms of this cascade with chronic stimulation, a murine model was created by overexpressing myocardial Gsα, a component of the βAR signaling pathway (1–7). Recent studies in our laboratory on this model demonstrated that cardiac Gsα overexpression enhances inotropic and chronotropic responses to endogenous sympathetic stimulation in younger animals, but as the animals age, a cardiomyopathy develops (2, 5). These studies of cardiac function were carried out using echocardiography in anesthetized mice. These in vivo techniques, however, are limited. The intrinsic regulation of LV myocyte inotropy by the βAR-Gs-AC pathway and the extent to which myocyte contraction and relaxation is altered by overexpression of Gsα, independent of the extracellular matrix and hemodynamic and neurohormonal effects, cannot be directly assessed. Furthermore, the prior in vivo studies of cardiac function in mice with overexpressed cardiac Gsα did not measure the effects on relaxation (2, 5).

In the current investigation, we have, to our knowledge characterized for the first time both contractile and relaxation function in isolated myocytes from mice with overexpressed cardiac Gsα. Although baseline contractile function was not altered in Gsα myocytes, consistent with previous in vivo observations in this model (2), myocyte contractile function was augmented in response to ISO and was associated with increased Ca²⁺ transients, assessed by fura loading, and with Ca²⁺ channel activity, assessed with patch-clamp measurements. Because forskolin, which stimulates cAMP distal to the βAR, elicited similar increases in contractile and relaxation function and Ca²⁺channel activity in both Gsα and WT mice, and, further, because forskolin’s action was blocked by Rp-cAMP, it can be concluded that altered AC catalytic activity was not the responsible mechanism. The results with CaCl₂ treatment, which increases inotropy independent of cAMP, and the lack of any observed differences between WT and Gsα myocytes in their contractile responses, also support the position that augmented inotropic responses to βAR stimulation with ISO in Gsα myocytes is a consequence of enhanced signaling via the βAR pathway rather than an alteration in Ca²⁺ handling or Ca²⁺ sensitivity at the subcellular-myofilament level.

We therefore attempted to determine whether the enhanced signal mediated by βAR stimulation in Gsα myocytes was due solely to increased cAMP production. To accomplish this, the effects of ISO stimulation were also examined by PKA blockade with Rp-cAMP. In WT myocytes, ISO no longer elicited an increase in systolic contraction after Rp-cAMP, indicating that essentially the entire response to ISO was cAMP-mediated. As already noted, the Gsα myocytes did not respond to forskolin with increased contraction after Rp-cAMP. In contrast, the Gsα myocytes still responded to ISO with enhanced contraction in the presence of Rp-cAMP. These experiments suggested that overexpressed Gsα permitted ISO to exert a positive inotropic effect independent of cAMP, e.g., potentially by an action directly or indirectly on the Ca²⁺ channel. In support of this hypothesis, measurement of I_Ca demonstrated a significantly increased response to ISO in the presence of Rp-cAMP in Gsα myocytes but not in WT myocytes. Interestingly, a recent study by Muntz et al. (6) demonstrated that the Gsα protein was localized in the T-tubules and intercalated disks in the Gsα myocytes. These and other data indicate that colocalization of the various components of both the βAR signaling unit (βAR-Gs-AC) and its targets, particularly those regulating Ca²⁺ handling, allows efficient and rapid activation of all components necessary to enhance contractility in response to βAR stimulation. Although Hartzell et al. (14) demonstrated that inotropic response to βAR stimulation was exclusively due to a cAMP-dependent pathway, other studies have identified another pathway by which βAR agonists can increase Ca²⁺ currents via a cAMP-independent pathway (10–13). The data in the present manuscript support the latter point of view (10–13) and thus may help to resolve this controversy.

The results of Ca²⁺ currents in this investigation differ in certain ways from a recent study by Lader et al. (7), which also used our Gsα myocytes but those that were dialyzed with the protein kinase inhibitor PKI and found to still exhibit an increase in I_Ca compared with nondialyzed Gsα myocytes. The study by Lader et al. found enhanced I_Ca at baseline in Gsα myocytes, which appears to be inconsistent with the current investigation. There is one important difference in the two studies: Lader et al. studied neonatal, cultured myocytes, whereas the current study used freshly prepared adult myocytes. Our preliminary data suggest that Gsα overexpression is enhanced in neonatal transgenic hearts compared with that in adult transgenic hearts (Vatner, D., unpublished data). Similarly, in neonatal myocytes from transgenic mice with β₂-AR overexpression, the baseline Ca²⁺ currents were also higher (31), but Ca²⁺ channel activity in adult β₂-AR–overexpressed myocytes is reduced compared with WT myocytes (32). Thus, the discrepancy between Ca²⁺ channel activity in neonatal and adult myocytes appears consistent for both β₂-AR overexpression and Gsα overexpression and points to an important limitation in extrapolating from neonatal or fetal to adult physiological regulation. This is particularly relevant to βAR-Gs-AC regulation, which is well recognized to be different in neonatal animals (15–17).

As already noted, prior studies in the Gsα model did not examine diastolic function. In the current investigation, echocardiographic assessment demonstrated enhanced relaxation in vivo, without an alteration in systolic contraction in overexpressed Gsα. The recovery of the late phase of diastole for both the myocyte length and fura signal was accelerated in the overexpressed Gsα myocytes at baseline. This suggests an interesting possibility that Ca²⁺ reuptake during late diastole is regulated by Gsα even at baseline. In further support of this possibility, after Rp-cAMP, the differences in recovery during late diastole were no longer different in Gsα and WT myocytes. The cellular basis for the accelerated relaxation in Gsα myocytes is not clear. It is possible that there is an enhanced SR Ca²⁺ uptake due to an increase in phosphorylation of phospholamban (33) in Gsα myocytes. Indeed, both increased cAMP levels at baseline and enhanced phospholamban phosphorylation were observed in the present study. It is also possible that other Ca²⁺ regulatory proteins are altered in this transgenic model, independent from, but potentially in response to, the actual genetic perturbation.

In conclusion, overexpression of Gsα resulted in more rapid relaxation at end diastole, but it does not affect baseline systolic function in isolated myocytes. Improved baseline diastolic function, independent from systolic function, was also observed in vivo. Both inotropic and lusitropic responses to βAR stimulation are enhanced in Gsα myocytes. The enhanced inotropic response to βAR stimulation partly reflects increased Ca²⁺ channel activity, and the cellular mechanisms mediating effects on both systolic and diastolic function appear to involve both a cAMP-independent as well as a cAMP-dependent pathway.

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