RhoA/ROCK Signaling and Pleiotropic α1A-Adrenergic Receptor Regulation of Cardiac Contractility

Aims To determine the mechanisms by which the α1A-adrenergic receptor (AR) regulates cardiac contractility. Background We reported previously that transgenic mice with cardiac-restricted α1A-AR overexpression (α1A-TG) exhibit enhanced contractility but not hypertrophy, despite evidence implicating this Gαq/11-coupled receptor in hypertrophy. Methods Contractility, calcium (Ca2+) kinetics and sensitivity, and contractile proteins were examined in cardiomyocytes, isolated hearts and skinned fibers from α1A-TG mice (170-fold overexpression) and their non-TG littermates (NTL) before and after α1A-AR agonist stimulation and blockade, angiotensin II (AngII), and Rho kinase (ROCK) inhibition. Results Hypercontractility without hypertrophy with α1A-AR overexpression is shown to result from increased intracellular Ca2+ release in response to agonist, augmenting the systolic amplitude of the intracellular Ca2+ concentration [Ca2+]i transient without changing resting [Ca2+]i. In the absence of agonist, however, α1A-AR overexpression reduced contractility despite unchanged [Ca2+]i. This hypocontractility is not due to heterologous desensitization: the contractile response to AngII, acting via its Gαq/11-coupled receptor, was unaltered. Rather, the hypocontractility is a pleiotropic signaling effect of the α1A-AR in the absence of agonist, inhibiting RhoA/ROCK activity, resulting in hypophosphorylation of both myosin phosphatase targeting subunit 1 (MYPT1) and cardiac myosin light chain 2 (cMLC2), reducing the Ca2+ sensitivity of the contractile machinery: all these effects were rapidly reversed by selective α1A-AR blockade. Critically, ROCK inhibition in normal hearts of NTLs without α1A-AR overexpression caused hypophosphorylation of both MYPT1 and cMLC2, and rapidly reduced basal contractility. Conclusions We report for the first time pleiotropic α1A-AR signaling and the physiological role of RhoA/ROCK signaling in maintaining contractility in the normal heart.


Introduction
Heart failure is a major cause of death, disability and escalating health costs worldwide as populations age. Inotropic drugs are useful short term, but their long term use may increase mortality, in part due to increased calcium (Ca 2+ ) release within cardiomyocytes (CMs). Sympathetic regulation of contractility reflects catecholamine stimulation of CM adrenergic receptors (ARs). b 1 -AR effects are normally dominant. In heart failure, however, b-ARs are downregulated and uncoupled from G proteins, and a 1 -ARs may act to maintain contractility.
We reported previously that transgenic mice with cardiacrestricted a 1A -AR overexpression (a 1A -TG) display hypercontrac-tility that is proportional to receptor number, is inhibited by selective a 1A -AR blockade, and is not due to b-AR cross-talk [1]. Surprisingly, this hypercontractility is not associated with cardiac hypertrophy [1,2]. Mice with 66-fold overexpression exhibit improved survival after pressure overload [3] or myocardial infarction [4], but 112-fold and 170-fold overexpression reduced survival due to sudden cardiac death consistent with Ca 2+ overload [5].
Here, we demonstrate that the mechanism underlying the enhanced cardiac contractility of a 1A -TG mice is indeed an agonist-induced increase in intracellular Ca 2+ release. In the absence of agonist, however, contractility is reduced. This unexpected finding was not due to reduced intracellular Ca 2+ concentration [Ca 2+ ] i but to reduced Ca 2+ sensitivity of the myofilaments resulting from inhibition of the RhoA/Rho kinase (ROCK) signaling pathway. This inhibition results in hypophosphorylation of myosin phosphatase target subunit 1 (MYPT1), relieving its inhibition of myosin light chain phosphatase (MLCP) and leading to inactivation of cardiac myosin light chain 2 (cMLC2), a key myofilament protein mediating contraction. The inhibition of RhoA/ROCK signaling was mediated by the a 1A -AR in the absence of ligand, consistent with spontaneous receptor isomerization to a conformation distinct from that which activates increased Ca 2+ release in the presence of ligand, indicating pleiotropic receptor signaling.
Critically, the regulation of basal contractility by the RhoA/ ROCK pathway is shown to be physiologically relevant because its inhibition in non-transgenic mice with normal receptor expression caused a significant reduction in basal contractile function. Because modulation of cMLC activity can increase contractility without altering [Ca 2+ ] i , the RhoA/ROCK signaling pathway may be a suitable target for development of novel inotropic interventions.

Animals
The a 1A -TG mice with cardiac-restricted a 1A -AR overexpression, established and bred with FVB/N, have been described in detail [1]. Notably, this model is based on overexpression of the wild type a 1A -AR, not a mutant, thus avoiding concerns of promiscuous activation of unrelated pathways. Male heterozygous a 1A -TG mice (170-fold overexpression) and their non-transgenic littermates (NTL) aged 8 to 10 weeks were used for this study. Experimental procedures were approved by the Garvan Institute of Medical Research/St Vincent's Hospital Animal Ethics Committee in accordance with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Calcium Sensitivity of Skinned Cardiac Fibers
Skinned left ventricular fiber strips were prepared as described previously [3]. Strips were skinned by immersion in 3% Triton X-100 for 30 min. Strips were activated with a series of solutions of increasing [Ca 2+ ] [3].

RhoA Activity
A G-LISA kit was used (BK124; Cytoskeleton Inc., CO). After lysis in a buffer containing 50 mM NaF (Sigma Aldrich), 20 mM sodium pyrophosphate (Sigma Aldrich) and 1 mM p-nitrophenyl phosphate (Merck Chemicals) to block phosphatase activity, homogenization, and protein concentration quantification, aliquots of lysate (0.5 mg protein/ml) were added to wells linked to a Rho-GTP-binding protein. Active (GTP-bound) RhoA attached to the wells. Inactive (GDP-bound) RhoA was eluted. Active RhoA was detected with an anti-RhoA antibody, and absorbance read at 490 nm with a PHERAStar FS microplate reader (BMG, Germany).

Statistical Analyses
All experiments and analyses were blinded. In skinned fibers, relative force was plotted against pCa (2log 10 [Ca 2+ ]) and fitted with Hill plots to determine pCa 50 (the pCa at half-maximal force) and Hill coefficient values [6]. In isolated hearts, the maximum rate of change of left ventricular pressure (dP/dt max ) was scaled as a % of baseline, and data from individual experiments fitted to a non-linear regression equation (Statistica programme, StatSoft, Tulsa, USA) to determine EC 25 , EC 50 and EC 75 , the agonist concentrations generating 25%, 50% and 75% of the maximum dP/dt max response, respectively, and analyzed using one-way ANOVA. Data points from different dose-response curves were compared using two-way ANOVA for repeated measures, with Newman-Keuls post-hoc test when indicated. For protein expression quantification, unpaired t tests were used for comparisons between two groups, and two-way ANOVA with multiple comparisons was used among groups (GraphPad Prism version 6.00 for Windows, GraphPad Software, California, USA). Results are mean 61 SEM.

CM Morphology
Cell morphology did not differ between a 1A -TG and NTL CMs ( Fig. 1A and Table 1 Kinetic studies showed no changes in Ca 2+ release or reuptake rates in a 1A -TG CMs (data not shown). a 1A -TG CMs are Hypersensitive to a 1A -AR Agonist Stimulation Despite the reduced shortening of a 1A -TG CMs observed in the absence of agonist stimulation, concentration-response curves to incremental doses of the a 1 -AR agonist, PE, demonstrated greater shortening of a 1A -TG than NTL CMs (Fig. 2C). The hypersensitivity of the shortening response to PE in a 1A -TG CMs paralleled increased Ca 2+ release with increasing agonist stimulation in these CMs, as reflected by the higher amplitude of the systolic rise in [Ca 2+ ] i (Fig. 2B). In contrast, NTL CMs did not demonstrate any significant change in either shortening or the amplitude of the [Ca 2+ ] i transient in response to PE. Resting [Ca 2+ ] i did not increase significantly with PE in a 1A -TG or NTL CMs ( Fig. 2A).  The isolated CM experiments suggested that the hypercontractility of a 1A -TG hearts in vivo might reflect hypersensitivity to endogenous catecholamines. To evaluate responses in the intact organ, we tested isolated perfused contracting heart preparations and found responses (Fig. 3) that closely mirrored those observed in isolated CMs. In the absence of agonist, isolated a 1A -TG hearts exhibited significantly reduced contractility (Fig. 3A), evidenced by lower peak pressure generation and lower dP/dt max , as well as impaired relaxation (dP/dt min ). Heart rate (a 1A -TG 381617 vs. NTL 371614 bpm) and coronary flow (2.560.2 vs. 2.260.1 ml/ min) were not different.
With the selective a 1A -AR agonist, A61603, the a 1A -TG hearts demonstrated marked hypercontractility, evidenced by higher peak pressure and higher dP/dt max for any given concentration of A61603, with parallel increments in dP/dt min (Fig. 3B, 3C). EC 50 was significantly lower in a 1A -TG hearts (0.08260.003 nM vs. NTL 10.162.8 nM, P,0.05). A61603 concentrations above 3 nM caused rapid decompensation in a 1A -TG hearts, corresponding to the sudden death phenotype documented previously in vivo [5].
Baseline Hypocontractility in a 1A -TG Hearts is not due to Heterologous Desensitization One possible explanation for baseline a 1A -TG hypocontractility is that sustained overstimulation of the contractile apparatus, due to activation of the greatly increased number of a 1A -ARs by endogenous catecholamines, results in heterologous downregulation of contractility at a sub-receptor level (that is, desensitization to multiple agonists resulting from excessive exposure to a single stimulus). If so, stimulation of the same sub-receptor pathway via an alternate Ga q -linked receptor would be expected to elicit a reduced  contractile response in a 1A -TG hearts. To test this hypothesis, isolated perfused hearts were treated with AngII to activate the Ga q/11 -coupled AT 1 receptor. AngII produced a transient negative, followed by a large sustained positive inotropic response (Fig. 4A24C). The positive inotropic effect of AngII, the increment in peak pressure or dP/dt max from baseline, was not reduced in a 1A -TG hearts (Fig. 4C).
Baseline Hypocontractility in a 1A -TG Hearts is Mediated by the a 1A -AR To test the alternative hypothesis that the hypocontractility observed in a 1A -TG hearts in the absence of agonist stimulation is mediated by the a 1A -AR, isolated perfused contracting hearts from a 1A -TG and NTL mice were perfused with the selective a 1A -AR antagonist, RS100329. RS100329 completely reversed the hypo- contractility and impaired relaxation observed in a 1A -TG hearts within 10 min of treatment onset (Fig. 4D, 4E). To ensure that this dramatic effect of RS100329 was indeed due to its specific antagonism of the a 1A -ARs, we repeated these experiments with a different selective a 1A -AR antagonist, KMD3213 dihydrobromide, and again demonstrated complete reversal of the hypocontractility and impaired relaxation observed in a 1A -TG hearts within 10 min of treatment onset (Fig. S1). Next we evaluated myofilament Ca 2+ sensitivity by measuring steady-state isometric force development in skinned cardiac fibers from a 1A -TG and NTL ventricular strips. Stepped changes in pCa produced increments in steady-state force, but the resulting composite Hill curves quantifying the force-Ca 2+ relationship were not significantly different for a 1A -TG and NTL ventricular strips (n = 13): pCa 50 5.8660.12 a 1A -TG vs. 5.8860.11 NTL; Hill coefficient 3.0360.12 nCa a 1A -TG vs. 2.8260.19 nCa NTL. The pCa 50 is a measure of the sensitivity of the contractile apparatus to Ca 2+ . The Hill coefficient gives an indication of the affinity of the functional unit of the contractile apparatus for Ca 2+ . a 1A -AR Overexpression Reduces Phosphorylation of cMLFC2 by Inhibiting RhoA Activity Although myofilament Ca 2+ sensitivity was not different in a 1A -TG skinned cardiac fibers, a modulating effect of myofilament protein phosphorylation on Ca 2+ sensitivity in vivo, where the cell membrane is intact, could not be excluded. To address this issue, we assessed the phosphorylation status of the key contractile proteins, cTnI and cMLC2, in isolated hearts from a 1A -TG and NTL mice perfused with vehicle (saline) or RS100329. There were no differences in total cTnI or p-cTnI, or in cTnC or cTnT, between a 1A -TG and NTL hearts (data not shown), but a 1A -TG mice exhibited significant hypophosphorylation of cMLC2 (Fig. 5A). Importantly, the decreases in p-cMLC2 and in the ratio of p-cMLC2/cMLC2 were rapidly reversed by the selective a 1A -AR antagonist, RS100329, within 8 minutes.
PKC, MLCK and MLCP regulate MLC phosphorylation in smooth muscle. PKCa was significantly higher in a 1A -TG (PKCa/ GAPDH 1.060.08) than NTL hearts (0.7160.08, P,0.05), but was not altered by RS100329 treatment. PKCe was unaffected by genotype or treatment (data not shown). MLCK phosphorylates MLC in response to changes in [Ca 2+ ] i , but was unaffected by genotype or RS100329 treatment (data not shown), consistent with the fact that baseline hypocontractility in a 1A -TG hearts was not associated with changes in [Ca 2+ ] i .
Conversely, dephosphorylation of MLC is mainly catalyzed by MYPT1, a myosin binding regulatory subunit of MLCP. The level of MYPT1 phosphorylated at Thr696, but not Thr853, was significantly reduced in a 1A -TG mice (Fig. 5B), and this was rapidly reversed by RS100329.
Given that active (that is, GTP-bound) RhoA binds to the Cterminal region of MYPT1, and that activated ROCK inhibits MLCP by phosphorylating MYPT1 at Thr696, we next examined RhoA/ROCK signaling. RhoA activity was significantly reduced in a 1A -TG hearts (Fig. 5B), a reduction rapidly reversed by RS100329, but protein expression of RhoA (Fig. 5A), or of ROCK1 or ROCK2 (data not shown), was unchanged. Cardiac contractility (dP/dt max ) was directly correlated with RhoA activity (R 2 = 0.85, Fig. 5C).

RhoA/ROCK Signaling Maintains Basal Cardiac Contractility in the Normal Heart
To further evaluate the involvement of RhoA/ROCK signaling in basal contractility, hearts were treated with Y-27632, a selective ROCK inhibitor. Selective ROCK inhibition caused significant falls in peak pressure, dP/dt max and dP/dt min in NTL hearts (Fig. 6A) within 5 minutes, accompanied by significant falls in the level of MYPT1 phosphorylated at Thr696 and p-cMLC2 (Fig. 6B), but caused no further reduction in basal contractility in a 1A -TG hearts, and had no effect on the increased contractility with A61603 in either NTL or a 1A -TG hearts (data not shown).

Mechanism of Increased Contractility without Hypertrophy with a 1A -AR Overexpression
We demonstrated that the mechanism of increased contractility with a 1A -AR overexpression was increased intracellular Ca 2+ release in response to agonist stimulation. This was not unexpected because other Ga q/11 -coupled receptors, including the AT 1 and endothelin receptors, increase Ca 2+ release by activating phospholipase Cb, and this would account for the increased PKCa expression we observed. In addition, a 1A -AR coupled Ca 2+ entry depends on a novel mechanism involving redirection and activation of the transient receptor potential canonical 6 (TRPC6) channel from the cytoplasm to the plasma membrane via interaction with Snapin, but a 1A -AR activation of Ga q/11 also produces diacylglycerol that independently activates TRPC6 in the plasma membrane [7]. Activation of the greatly increased number of a 1A -ARs by endogenous catecholamines could thus account for the hypercontractility seen in vivo [1], but the elevated [Ca 2+ ] i might be expected to stimulate cardiac hypertrophy also. Marked hypertrophy is observed, for example, in mice with cardiac overexpression of Ga q [8] or other Ga q/11 -coupled receptors, such as the AT 1 receptor [9], yet hypertrophy was not evident in our a 1A -TG CMs or in mouse or rat hearts in vivo [1,10]. This may be because a 1A -AR activation in vivo is not sustained but intermittent, fluctuating with endogenous catecholamine levels. This is consistent with the propensity of a 1A -TG mice to stress-related sudden cardiac death suggestive of Ca 2+ overload [5]. Sustained a 1A -AR activation would be expected to cause heterologous desensitization of the contractile response [11], but we found no evidence of this. Despite the large increase in the systolic amplitude of the [Ca 2+ ] i transient with agonist stimulation of a 1A -TG CMs, we observed no change in resting [Ca 2+ ] i with repeated but non-sustained a 1A -AR activation (Fig. 2C), which could account for the lack of hypertrophy.

Mechanism of Reduced Contractility with a 1A -AR Overexpression in the Absence of Agonist
The unexpected finding in our study was the reduced contractility observed with a 1A -overexpression in the absence of agonist. Overexpression of other G protein-coupled receptors, such as the b-AR, results in marked agonist-independent receptor signaling due to spontaneous receptor isomerization [12]. The hypocontractility with a 1A -AR overexpression was not due to any alteration in [Ca 2+ ] i . Nor was the hypocontractility due to heterologous desensitization, as noted above. We also demonstrated that the sensitivity of the contractile machinery to Ca 2+ was unaltered in a 1A -TG skinned cardiac fibers, but this preparation is minimally phosphorylated [13].
We explored whether myofilament Ca 2+ sensitivity was impaired due to altered phosphorylation. In cardiac muscle, Ca 2+ sensitivity is thought to be regulated mainly by the troponin complex, but we found no alterations in the cardiac troponins or their phosphorylation status. In smooth muscle, contraction is primarily dependent on phosphorylation of regulatory MLC, which is controlled by the opposing activities of Ca 2+ /calmodulindependent MLCK and Ca 2+ -independent MLCP. Moreover, activation of the small GTPase, RhoA, and its downstream target, ROCK, results in Ca 2+ sensitization as a result of MYPT1 phosphorylation and, thus, inhibition of MLCP, increasing MLC phosphorylation in smooth muscle [14]. Phosphorylated MLC binds to myosin at the head-rod junction, which facilitates actinmyosin interactions that enhance contractility.
Our major finding was that the reduced cardiac contractility with a 1A -TG overexpression was due to cMLC2 hypophosphorylation. We explored whether this was driven by alterations in MLCK or the RhoA/ROCK signaling pathway. Because there was no change in [Ca 2+ ] i , the absence of any change in expression of the Ca 2+ /calmodulin-dependent MLCK was expected. The significant hypophosphorylation of cMLC2 was due to reduced RhoA activity and reduced phosphorylation of MYPT1. RhoA activity was strongly correlated with cardiac contractility. Importantly, the hypocontractility and all of the changes in the RhoA/ ROCK signaling pathway were rapidly reversed by selective a 1A -AR blockade. In contrast, the increased PKCa expression we observed in a 1A -TG hearts, which could conceivably have contributed to the hypocontractility [15], was unchanged with selective a 1A -AR blockade.

Pleiotropic Signaling by the a 1A -AR
The rapid reversal of the agonist-independent hypocontractility in a 1A -TG hearts after selective a 1A -AR blockade with two different selective antagonists indicates that the hypocontractility results from spontaneous receptor activity. But the activated states in the absence and presence of agonist are different: hypocontractility in the absence but hypercontractility in the presence of agonist. These effects cannot be explained by promiscuous coupling to extraneous pathways as a result of a 1A -AR overexpression because the a 1A -AR used to develop the a 1A -TG model was the wild type, not a mutant [1].
We propose a model of pleiotropic receptor signaling (Fig. 7) in which contractility is suppressed by engagement of the agonistindependent activated conformation of the receptor (R*) with the RhoA/ROCK pathway, leading to its inhibition. In contrast, agonist activation of the receptor induces a distinct active conformation (R**) that does not involve engagement of the RhoA/ROCK pathway but enhances contractility by both a 1A -AR coupled Ca 2+ entry [7] and Ga q/11 -dependent Ca 2+ release. We have shown previously that a single receptor subtype can adopt differing activated conformations to engage distinct downstream signaling pathways [16,17]. How R* suppresses RhoA/ROCK signaling is presently being investigated, but the rapid reversal after selective a 1A -AR blockade points to altered protein activation rather than expression. Potential mechanisms include activation of a RhoA guanine nucleotide dissociation inhibitor (RhoGDI) [18], either directly or by initial interaction of R* with a b-arrestin, perhaps by activating a kinase that phosphorylates RhoGDI, or inhibits a GDI displacement factor that mediates RhoA.RhoGDI dissociation.

Physiological Role of RhoA/ROCK Signaling and Clinical Implications
The link between cardiac contractility and RhoA/ROCK signaling in animals with 170-fold overexpression of the a 1A -AR raises the question of physiological relevance. Although contrac-tility is reduced in mice with a non-phosphorylatable form of cMLC2, and reduced phosphorylation of cMLC2 has been found in failing human and mouse hearts [19,20], a physiological role for cMLC2 in regulating cardiac contractility has not been clearly established. Similarly, chronic inhibition of the RhoA/ROCK pathway may prevent adverse remodeling in experimental heart failure models [21,22], but its physiological role in regulating contractility remains unclear. ROCK inhibition has been reported to decrease endothelin-1 induced increases in contractility in rabbit ventricular CMs [23], but others have reported enhanced cardiac contractility after ROCK inhibition in infarct and diabetic experimental models [24,25].
To address this issue more directly, we examined ROCK inhibition in NTL hearts with normal a 1A -AR expression, demonstrating a significant reduction in baseline contractility in association with reduced phosphorylation of MYPT1 and cMLC2. These findings indicate that the RhoA/ROCK pathway plays an important physiological role in maintaining normal baseline contractility. This normal role may be amplified in heart failure, when the b-ARs are downregulated and uncoupled from G proteins, and with the increasing therapeutic use of b-AR blockers. Moreover, increased contractility with RhoA/ROCK pathway activation does not depend on increased Ca 2+ release, suggesting it as a promising target for development of novel inotropic agents that might not increase mortality with long term use.

Limitations
Our novel finding of depressed cardiac contractility due to agonist-independent activity of the a 1A -AR is based on a model with 170-fold overexpression of the receptor. Nevertheless, this model has allowed us to identify pleiotropic signaling by the receptor that may have broader significance for receptor physiology. As noted above, promiscuous coupling due to receptor overexpression can be excluded because the model is based on the wild type a 1A -AR. This model also allowed us to identify the importance of RhoA/ROCK signaling and its control of MLC2 phosphorylation in modulating cardiac contractility, and we have demonstrated that this mechanism supports baseline contractility even in the setting of normal a 1A -AR expression. The mechanism by which the a 1A -AR inhibits RhoA activity in the absence of ligand remains to be determined in future experiments. Figure S1 Hypocontractility and impaired relaxation in a 1A -TG hearts are reversed with the selective a 1A -AR antagtonist, KMD3213. A, representative recordings of left ventricular pressure (LVP) and dP/dt at baseline and during KMD3213 infusion (100 nM) in isolated perfused contracting hearts. B, composite data at baseline (Control) and after KMD3213 infusion (100 nM) for 10 min in NTL (n = 4) and a 1A -TG (n = 4) hearts. Data are shown as the mean 6 SEM. *P, 0.05, **P,0.01.