β₁-Adrenergic receptors stimulate cardiac contractility and CaMKII activation in vivo and enhance cardiac dysfunction following myocardial infarction

Abstract

The β-adrenergic receptor (βAR) signaling system is one of the most powerful regulators of cardiac function and a key regulator of Ca²⁺ homeostasis. We investigated the role of βAR stimulation in augmenting cardiac function and its role in the activation of Ca²⁺/calmodulin-dependent kinase II (CaMKII) using various βAR knockouts (KO) including β₁ARKO, β₂ARKO, and β₁/β₂AR double-KO (DKO) mice. We employed a murine model of left anterior descending coronary artery ligation to examine the differential contributions of specific βAR subtypes in the activation of CaMKII in vivo in failing myocardium. Cardiac inotropy, chronotropy, and CaMKII activity following short-term isoproterenol stimulation were significantly attenuated in β₁ARKO and DKO compared with either the β₂ARKO or wild-type (WT) mice, indicating that β₁ARs are required for catecholamine-induced increases in contractility and CaMKII activity. Eight weeks after myocardial infarction (MI), β₁ARKO and DKO mice showed a significant attenuation in fractional shortening compared with either the β₂ARKO or WT mice. CaMKII activity after MI was significantly increased only in the β₂ARKO and WT hearts and not in the β₁ARKO and DKO hearts. The border zone of the infarct in the β₂ARKO and WT hearts demonstrated significantly increased apoptosis by TUNEL staining compared with the β₁ARKO and DKO hearts. Taken together, these data show that cardiac function and CaMKII activity are mediated almost exclusively by the β₁AR. Moreover, it appears that β₁AR signaling is detrimental to cardiac function following MI, possibly through activation of CaMKII.

β-adrenergic receptors (βARs) play a critical role in the maintenance of cardiac homeostasis (10). Regulation of these homeostatic mechanisms may in part be due to differences in the signaling pathways that are activated by the two major βAR subtypes in the heart (11, 49). Although both β₁ and β₂AR subtypes can couple to G_s proteins to activate adenylyl cyclase and increase cAMP levels (41), β₂ARs are also able to couple to G_i, which may promote cell survival signals and protect the heart during heightened sympathetic nervous system activity (23, 48, 54). However, to what degree β₁AR promote detrimental signaling to cause pathological cardiac dysfunction is not well understood.

Evidence to support a pathogenic role for β₁ARs in heart failure comes from studies showing that overexpression of β₁ARs in mice causes early hypertrophy and interstitial fibrosis followed by marked cardiac dysfunction (18). In contrast, modest overexpression of β₂ARs (60- to 100-fold over endogenous levels) results in enhancement of cardiac function without deterioration of cardiac function (30, 34). Moreover, in isolated myocytes, β₂AR stimulation protects against stress-induced apoptosis (12, 51), whereas in adult mice lacking β₂ARs chronic isoproterenol (Iso) stimulation results in enhanced myocyte apoptosis and increased mortality compared with mice lacking β₁ARs (39). In addition, in a rodent model of myocardial infarction (MI)-induced heart failure, selective activation with a β₂AR agonist improves cardiac performance without promoting apoptosis (1). Although these data support a concept that β₂AR signaling is cardioprotective, it has been suggested that opposing the protective effect of β₂AR signaling is β₁AR mediated activation of Ca²⁺/calmodulin-dependent kinase II (CaMKII) (55).

CaMKII belongs to a family of multifunctional protein kinases that regulate Ca²⁺ homeostasis and are essential for normal cardiac function (5, 7, 9). Enhanced βAR stimulation has been shown to activate apoptotic signaling pathways via PKA-independent stimulation of L-type Ca²⁺ channels, resulting in the activation of CaMKII (7, 55), which may participate in the deterioration of cardiac function in the failing heart (52). Precisely which βAR subtype is responsible for activation of CaMKII in the heart has not been fully elucidated.

To test the individual contribution of β₁ and β₂ARs in determining normal left ventricular (LV) function and whether βAR subtypes are important for the development of cardiac dysfunction, we studied knockout (KO) mice lacking either β₁ARs (β₁AR knockout, β₁ARKO), β₂AR (β₂AR knockout, β₂ARKO) or both β₁ARs and β₂ARs (β₂AR, and β₂AR knockouts, DKO). In this study we identify the specific contribution of the β₁AR on LV contractile function and CaMKII activation and the role the β₁AR plays in the development of cardiac dysfunction following MI.

METHODS

Experimental animals and surgical preparation.

The animals in this study were handled according to animal welfare regulations and protocols approved by the authors' Institutional Review Boards. Genetically engineered, 8- to 12-wk-old β₁ARKO, β₂ARKO, DKO, and C57/B6 wild-type (WT) mice were used for this study (43). Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg). Under a dissecting microscope Nissho Optical T-240 (Labtek, Campbell, CA) animals were placed in the supine position on a heated operation board and a midline cervical incision was made to expose the trachea. Following successful endotracheal intubation, the cannula was connected to a rodent ventilator. At study termination, hearts were removed and chambers were separated and weighed. The entire LV (infarct and noninfarct zone) was divided in half between the base and apex and used for histology and biochemical assays, respectively.

Hemodynamic measurement and P-V loops.

In vivo pressure-volume (P-V) analysis was performed as previously described (19, 40). Briefly, after bilateral vagotomy, the chest was opened and the pericardium was dissected to expose the heart. A 7-0 suture ligature was placed around the transverse aorta to manipulate loading conditions. A 1.4-Fr pressure-conductance catheter (Millar Instruments, Houston, TX) was inserted into the right carotid artery and advanced retrograde into the LV to record hemodynamics. A polyethylene-50 catheter was inserted into the right external jugular vein for Iso infusion. Steady-state pressure and volume measurements were recorded at baseline and after 3-min Iso infusion (20 pg·g⁻¹·min⁻¹). Pressure and volume measurements were obtained during the increase in the afterload generated by gently pulling on the suture to transiently constrict the aorta. Subsequently, parallel conductance (Vp) was determined by 10-μl injection of 15% saline into the right jugular vein to establish the offset due to the conductivity of structure to the blood pool. The derived Vp was used to correct the P-V loop data. Data were recorded digitally at 1,000 Hz and analyzed with pressure volume analysis software (PVAN data analysis software version 3.3; Millar Instruments) as previously described (40).

Western blotting analysis of CaMKII activation and PLB phosphorylation.

To evaluate the activation of CaMKII and phospholamban (PLB) phosphorylation, LV tissue was homogenized in Nonidet P-40 lysis buffer containing 20 mM Tris (pH 7.4), 137 mM NaCl, 1% Nonidet P-40, 20% glycerol, 10 mM PMSF, 1 mM Na₃VO₄, 10 mM NaF, 2.5 μg/ml aprotinin, and 2.5 μg/ml leupeptin. Protein concentration of sample was assayed with Bio-Rad protein assay reagent, and 100 μg of protein was denatured by heating at 95°C for 5 min prior to resolution by SDS-PAGE. Total CaMKII and pCaMKII (autophosphorylation site at Thr-286) were detected by using anti-CaMKII (1:2,000, Santa Cruz) and anti-phosphorylated CaMKII at Thr-286 (1:2,000, Cell Signaling), respectively. PLB phosphorylation was detected with the phosphorylation site-specific antibodies recognizing phospho-PLB (Ser-16) or phospho-PLB (Thr-17) (1:2,000, Santa Cruz). Total PLB was detected with polyclonal antibody against PLB (1:2,000, Santa Cruz). Immunoblots were revealed by enhanced chemiluminescence (ECL, Amersham Biosciences).

Myocardial infarction.

Myocardial infarction was performed as previously described (15). A left thoracotomy was performed in the fourth intercostals space at the left sternal border and MI was produced by ligation the left anterior descending (LAD) coronary artery with an 8-0 prolene suture at the site of the vessels' emergence past the tip of the left atrium. The chest cavity was closed, the mouse was gradually weaned off the respirator, and the endotracheal tube was removed. The animal was placed in a cage on a heating pad until fully conscious.

Serial echocardiography.

Transthoracic echocardiography was performed with a linear 30-MHz transducer (Vevo 770 High Resolution Imaging System, VisualSonics, Toronto, ON, Canada). LV end-diastolic and end-systolic dimensions (LVEDD and LVESD, respectively), fractional shortening (FS), interventricular septal wall thickness, posterior wall thickness, and heart rate were acquired in conscious mice placed on a heated pad as previously described (20, 35, 40).

CaMKII activity assay.

The enzyme activity was assayed in a total assay volume of 50 μl with use of a CaMKII peptide (281-291) as the enzyme substrate. The assay contained 50 mM Tris·HCl (pH 7.4), 382.7 μM CaMKII substrate (281-291, Calbiochem), 0.3 mM ATP ([γ^-32P]ATP = 1 μCi per assay), 10 mM magnesium acetate, 0.2 mM EDTA, ±5 μM calmodulin, ±250 μM CaC1₂ and an indicated amount of tissue fraction with varying protein concentrations from 0 to 70 μg. The reaction was initiated by adding enzyme, incubated at 30°C for 10 min, and terminated by adding 20 μl of 300 mM. EDTA and ³²P incorporation in the substrate were determined as previously described (24).

Histological analysis and determination of infarct size.

After termination of the experiment, hearts were excised and the LV was transversely dissected into two halves. The portion from the base to the midcavity was flash frozen and stored for subsequent biochemical studies. The portion of LV from midcavity to apex was fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with Masson's trichrome. Infarct size was determined by studying serial sections and calculating the mean percentage of epicardial and endocardial circumference that was scar as previously described (15).

βAR density.

Myocardial membranes were prepared by homogenization of the base of the LV as described (35, 40). Total βAR density was determined by incubation of 20 μg of membranes with a saturating concentration (200 pmol/l) of ¹²⁵I-labeled cyanopindolol and 20 mmol/l alprenolol to define nonspecific binding. Assays were conducted at 37°C for 60 min and then filtered over GFIC glass fiber filters (Whatman), then washed and counted in a gamma counter as previously described (35, 40).

In situ TUNEL staining.

Heart specimens were fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5-μm thickness. DNA fragmentation was detected in situ in deparaffinized sections using the ApopTag Kit (Intergene) according to manufacturer's protocol as previously described (35). The total number of nuclei was determined by manual counting of DAPI-stained nuclei in six random fields per section (magnification, ×200). All terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive nuclei were counted in each section.

Statistical analysis.

Values are shown as means ± SE. The significance of differences between baseline and Iso stimulation within same genetic species and between Pre (before MI) and 8-wk post-MI echocardiographic values within group was evaluated by Student's t-test. To evaluate differences hemodynamic parameters among groups from P-V loops, multigroup comparisons were made by use of a one-way ANOVA and Tukey's test. Also, ANOVAs with Bonferroni correction for multiple comparisons were used to evaluate the morphometric and serial echocardiographic measurements and CaMKII activity. P < 0.05 was considered significant.

RESULTS

Catecholamine stimulation of cardiac inotropy and chronotropy is mediated by β₁ARs.

To test which βAR subtype is responsible for the inotropic and chronotropic response of the heart, we administered Iso to the β₁ARKO, β₂ARKO, DKO, and WT mice and measured contractile function by P-V loop analysis. Basal ejection fraction as measured by PV loops and peak rate of pressure rise (dP/dt_max) was similar among the different genotypes (Table 1, Fig. 1). In contrast, in response to Iso only the WT and β₂AR KO mice showed an increase in the slope of the end-systolic P-V relation, indicating increased contractility, whereas the β₁AR KO and DKO showed no significant response to catecholamine infusion (Table 1, Fig. 1). Thus the presence of β₁ARs are necessary for an Iso induced increase in heart rate, dP/dt_max, maximal power, and end-systolic elastance by 40–65%, with no response observed to Iso infusion in the β₁ARKO and DKO mice (Fig. 2). No changes in diastolic parameters are found for the different groups.

Table 1.

Hemodynamic parameters in anesthetized instrumented wild-type and βAR KO mice

	Wild Type (n = 8)		β1ARKO (n = 9)		β2ARKO (n = 8)		β1 and β2AR DKO (n = 8)
	Base	Iso	Base	Iso	Base	Iso	Base	Iso
	Basic parameters
BW, g	32.1±1.3		28.1±2.7		28.8±2.9		25.7±1.3
HR, beats/min	438±28	599±19†	410±12	400±9§	367±10	539±17†	425±21	424±260§
ESP, mmHg	91.3±2.7	93.4±2.9	84.7±2.9	84.1±1.6	89.5±1.7	87.2±3.3	83.2±4.3	94.4±3.3
EDP, mmHg	11.3±0.7	7.7±1.6	8.1±1.4	8.6±1.7	6.0±1.1	6.3±1.0	8.9±23.8	12.3±4.7
ESV, μl	12.3±2.0	6.0±1.2*	10.0±1.7	9.5±1.7	12.2±2.4	9.5±1.5	6.3±2.8	9.4±3.5
EDV, μl	28.7±2.9	25.0±3.0	28.5±2.5	28.0±2.9	25.5±3.3	22.9±2.4	20.0±1.6	23.5±0.9
Stroke volume, μl	18.8±1.1	20.5±2.3	22.1±2.3	22.1±2.6	17.1±1.5	16.7±2.0	17.1±1.3	18.1±1.2
	Systolic function
EF, %	65.6±4.6	80.1±4.4*	70.7±2.8	71.0±2.8	59.6±4.0	65.6±5.1	70.6±4.8	62.4±5.5‡
dP/dtmax, mmHg/s	8,176±629	13,082±506‡	7,989±782	7,447±781§	6,658±544	11,309±997†	8,510±908	9,214±944§
Max power, mW	7.35±0.18	11.84±0.73†	7.91±0.94	7.87±0.92‡	6.80±0.59	10.47±0.93†	6.88±0.35	6.95±0.33§
Ees, mmHg/μl	6.71±0.30	11.1±1.07†	8.46±0.60	7.34±0.80‡	6.90±0.77	10.09±0.80*	9.49±1.60	7.66±1.71
	Diastolic function
dP/dtmin, −mmHg/s	5602±487	6490±465	3939±459	3825±512§	4340±351	5550±486	4445±472	5485±490
Tau (τ), ms	15.6±0.8	14.0±0.6	18.8±2.0	17.2±1.7	20.7±1.4	16.7±1.7	21.8±2.2	20.6±3.6
EDPVR, mmHg/μl	0.57±0.10	0.32±0.12	0.48±0.10	0.54±0.30	0.40±0.09	0.23±0.27	0.47±0.21	0.72±0.21

All values are expressed as means ± SE. βAR, β−adrenergic receptor; KO, knockout; Base, baseline; Iso, isoproterenol; β₁ARKO, β₁-adrenergic receptor knockout; β₂ARKO, β₂-adrenergic receptor knockout; DKO, double knockout; BW, body weight; HR, heart rate; ESP, left ventricular end-systolic pressure; EDP, end-diastolic pressure; ESV, end-systolic volume; EDV, end-diastolic volume; EF, ejection fraction; dP/dt_max, peak rate of pressure rise; −dP/dt_min, peak rate of pressure decline; Max power; maximum value of power during cardiac cycle (mW); E_es, end-systolic elastance (slope of the end-systolic relationship); Tau(τ), relaxation time constant calculated by Glantz method (regression of dP/dt vs. pressure); EDPVR: end-diastolic elastance (slope of the end-diastolic relationship).

^*P < 0.05,

^†P < 0.005 vs. base of same genotype;

^‡P < 0.05,

^§P < 0.005 either base in WT vs. base in KO mice or Iso in WT vs. Iso in KO mice.

Fig. 1.

Representative case of basic hemodynamic parameters (A–D) and pressure-volume (P-V) loop with end-systolic elastance (E_es) in mice. Basal heart rate (HR) and peak rise in pressure (dP/dt_max) are markedly increased following isoproterenol (Iso) infusion in wild-type (WT; A) and β₂-adrenergic receptor knockout (β₂ARKO; C) mice. There is no increase in β₁ARKO (B) or double knockout (DKO; D) mice. The P-V relationships display no change after isoproterenol in mice lacking β₁ARs (i.e., β₁ARKO and DKO; G and H), whereas contractility is increased in the WT (E) and β₂ARKO mice (F).

Fig. 2.

Hemodynamic changes (%) of baseline value after isoproterenol stimulation in mice (n = 8–9 for each group). In WT and β₂ARKO mice, heart rate, dP/dt_max, maximal power, and E_es are significantly increased compared with basal levels, with no significant increase seen in β₁AR or DKO mice (A–D). *P < 0.005 WT or β₂AR vs. β₁AR or DKO, #P < 0.05 WT or β₂AR vs. β₁AR or DKO.

Stimulation of β₁AR activates CaMKII signaling pathway.

To determine the βAR subtype in the heart that is responsible for the activation of CaMKII, we measured the extent of CaMKII phosphorylation on the autophosphorylation site Thr-286 in hearts from the different βARKO mice. After 5 min of Iso infusion, CaMKII activity significantly increased in WT and β₂AR KO as shown by the increased phosphorylation of the cytoplasmic CaMKIIδC isoform (Fig. 3A). In contrast, stimulation with Iso in β₁ARKO and DKO mice did not affect CaMKII phosphorylation in the heart (Fig. 3A). To determine whether the differential activation of CaMKII by the βAR subtypes is translated to downstream signaling pathways, we measured the level of phosphorylation of the sarcoplasmic reticulum protein PLB, a known effector of PKA and CaMKII signaling (52).Since PKA and CaMKII can independently phosphorylate PLB at Ser-16 and Thr-17, respectively, we examined the site-specific phosphorylation of PLB in response of βAR stimulation. Following Iso infusion, phosphorylation at both Ser-16 (PKA site) and Thr-17 (CaMKII site) were significantly increased only in WT and β₂ARKO mice and were unchanged from basal levels in the β₁ARKO and DKO mice (Fig. 3B). These data show that in the in vivo heart CaMKII activation and the subsequent enhancement of downstream signaling are mediated solely by stimulation of β₁AR.

Fig. 3.

Isoproterenol-stimulated Ca²⁺/calmodulin-dependent kinase II (CaMKII; A) and phospholamban (PLB; B) activity in mice (n = 4 for each group). A: representative immunoblot showing accumulation of phospho-CaMKII after treatment with 10 μg·g⁻¹·min⁻¹ isoproterenol infusion in WT and β₂ARKO mice. Summary graph represents a significant increase of phospho-CaMKII in WT and β₂ARKO mice. B: representative immunoblot showing accumulation of phospho-PLB after treatment with 10 μg·g⁻¹·min⁻¹ isoproterenol infusion in WT and β₂ARKO mice. Summary graph represents a significant increase of Ser-16 (left) and Thr-17 (right) in WT and β₂ARKO mice. *P < 0.005, treatment with isoproterenol vs. without isoproterenol for each group.

Mice lacking β₁ARs have preserved cardiac function after MI.

To determine whether the two βAR subtypes play a differential role in the development of postinfarct cardiac dysfunction, we subjected 12-wk-old β₁ARKO, β₂ARKO, DKO, and WT mice to MI by ligation of the LAD coronary artery. Serial echocardiographic measurements were obtained in the WT and the various βARKO mice prior to LAD ligation and at 4 and 8 wk post-MI. Histological analysis of myocardial scar via Masson's trichrome staining revealed similar infarct size for all four groups (WT: 33 ± 3%, β₁ARKO: 29 ± 3%, β₂ARKO: 29 ± 2%, and DKO: 33 ± 3%, P = not significant, Fig. 4A). Despite the similar infarct size between the four groups, β₁AR, β₂AR, DKO, and WT mice showed differing severity in the subsequent development of cardiac dysfunction as demonstrated by M-mode echocardiography (Fig. 4B, Table 2). At baseline, β₁ARKO and DKO mice showed reduced cardiac function compared with either WT or β₂ARKO mice, reflecting the contribution of β₁ARs to the maintenance of normal cardiac function (FS of 47 and 45% for β₁ARKO and DKO, respectively, vs. 64% for both β₂ARKO and WT mice). Eight weeks post-MI, WT and β₂ARKO mice showed progressive LV enlargement as measured by the percent change in LVEDD and LVESD associated with significant fall in %FS (Fig. 4, C–E). In contrast, both β₁ARKO and DKO mice showed an attenuated decline in cardiac function after MI as demonstrated by smaller changes in % FS and LV dimensions compared with preinfarction (Fig. 4, C–E).

Fig. 4.

β₁ARs promote detrimental cardiac remodeling in WT and β₂AR KO mice post-MI. A: histological sections of infarcted hearts are shown at 8 wk post-MI. B: representative M-mode echocardiograms of WT and KO mice pre-MI (Pre) and at 4 wk (4W) and 8 wk (8W) post-MI. C–E: serial echocardiography in conscious WT and βARKO mice after MI. C: % left ventricular end-diastolic dimension (LVEDD) change. D: % left ventricular end-systolic dimension (LVESD) change. E: % fractional shortening (FS) change. *P < 0.005 β₁AR (n = 13) or DKO (n = 15) vs. β₂AR (n = 16) or WT (n = 18) at 8 wk. †P < 0.005 β₁AR vs. WT, β₂AR, or DKO at 4 wk.

Table 2.

Morphometric and echocardiographic parameters in conscious mice pre and 8 wk post-MI

	Wild type (n = 18)		β1AR KO (n = 13)		β2AR KO (n = 16)		β1 and β2AR KO (n = 15)
	Pre	8W	Pre	8W	Pre	8W	Pre	8W
BW, g	25.7±0.9	27.5±0.9	30.6±1.6	31.8±1.6	29.1±1.7	33.8±1.5	29.3±1.4	30.8±1.4
LVW, mg		131.8±6.5		107.4±6.3		119.3±8.3		108.5±6.5
RVW, mg		30.8±2.7		29.5±0.9		25.5±2.4		25.10±1.0
LVW/BW, mg/g		4.9±0.3‡		3.5±0.3		3.6±0.2		3.5±0.1
IS%,		32.5±2.8		29.3±3.2		28.5±2.4		29.6±2.7
LVEDD, mm	2.9±0.06	4.5±0.2*	3.5±0.1	4.4±0.2*	2.8±0.06	4.3±0.1†	3.4±0.1	4.4±0.2*
LVESD, mm	1.1±0.05	2.8±0.2*	1.9±0.1	2.7±0.2*	1.0±0.06	2.7±0.1†	1.9±0.1	3.0±0.2*
IVSWth, mm	0.8±0.05	0.7±0.1	0.8±0.04*	0.6±0.05	1.0±0.03	0.7±0.1*	0.7±0.1	0.5±0.04
PWth, mm	0.8±0.06	0.8±0.1	0.9±0.06	0.8±0.07	1.0±0.02	0.8±0.1	0.8±0.03	0.6±0.04
FS, %	64.0±1.0	39.0±1.0†	47.0±1.0§	39.0±3.0*	64.0±2.0	37.0±2.0†	45.0±1.0§	33.0±1.0*
HR, bpm	577±30	659±18	485±14	393±31§	600±25	593±30	496±17	493±12

All values are expressed as means ± SE. Pre, before myocardial infarction; 8W, 8 wk after myocardial infarction; LVW, left ventricular weight; RVW, right ventricular weight; IS, infarct size; LVEDD, left ventricular end-diastolic dimension; LVESD, left venticular end-systolic dimension; IVSWth, interventricular septal wall thickness; PWth, posterior wall thickness; FS, fractional shortening.

^*P < 0.005,

^†P < 0.0001 8W vs. Pre within group;

^‡P < 0.05 LVW/BW WT vs. β1ARKO, β2ARKO, DKO; HR 8W vs. Pre for β1ARKO;

^§P < 0.05 Pre in WT vs. Pre in KO mice or 8W in WT vs. 8W in KO mice.

Downregulation of βARs only in the WT and β₂ARKO mice following MI.

Since downregulation of βARs is a hallmark of failing myocardium, we measured βAR density in sham and MI hearts from the four groups of mice. Sham WT and sham β₂ARKO hearts had similar βAR densities (40 and 35 fmol/mg protein, respectively) consistent with the known data that the majority of βARs in the myocardium are of the β₁AR subtype (Fig. 5A). Following MI, βAR downregulation was observed only in the WT and β₂ARKO mice, demonstrating that β₁AR is the primary receptor subtype that undergoes downregulation in the failing heart (Fig. 5A).

Fig. 5.

βAR density and CaMKII activity following MI in βAR knockout mice (n = 5 for each group). A: only β₁ARs are downregulated 8 wk post-MI, whereas receptor densities of β₂ARs remain unchanged. *P < 0.05 sham vs. MI within group. B: CaMKII activity post-MI. C: sham hearts. The presence of β₁AR stimulates Ca²⁺-dependent CaMKII activity after MI. *P < 0.05 Ca²⁺ dependent vs. Ca²⁺ independent within group.

Presence of β₁AR contributes to elevated CaMKII activity following MI.

CaMKII is a downstream target of β₁AR signaling and recent studies have shown that CaMKII inhibition can substantially reduce myocyte apoptosis and maladaptive remodeling from excessive βAR activation (52, 55). To examine whether in the failing heart the activation of CaMKII was dependent on a specific βAR subtype, we evaluated CaMKII activity in each subgroup after either sham surgery or MI. A significant increase in Ca²⁺-dependent CaMKII activity was seen in WT and β₂ARKO hearts 8 wk post-MI (Fig. 5B). In contrast, no increase in CaMKII activity was found in the β₁ARKO and DKO post-MI hearts (Fig. 5B). As expected, there were no differences in Ca²⁺-dependent CaMKII activity in the sham-operated controls (Fig. 5C). As a positive control, Iso infusion in normal WT mice significantly increased CaMKII activity (Fig. 5, B and C).

Increased apoptosis in WT and β₂ARKO mice within the infarct border zone following MI.

To test whether divergent signals by the individual βAR subtypes would contribute to differences in the development of apoptosis, TUNEL staining was performed within the border zone and remote regions from the infarcted tissue. The number of TUNEL-positive nuclei was significantly higher within the border zone of the infarct in the β₂ARKO and WT hearts compared with that in the β₁ARKO and DKO hearts (P < 0.004) (Fig. 6A). TUNEL staining in the remote zones distant from the infarcted tissue showed no differences in the level of apoptosis between the groups (Fig. 6B).

Fig. 6.

β₁ARs promote apoptotic cell death after MI (n = 6 for each group). A: border zone. B: remote zone. Increased terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive nuclei were identified in WT and β₂AR KO mice after 8 wk MI in border zone. No difference in rate of apoptosis at remote zone. *P < 0.05 WT or β₂AR vs. β₁AR or DKO.

DISCUSSION

In our present study using specific βARKO mice, we demonstrate that the β₁AR specifically contributes to the increase in LV function and CaMKII activation following acute adrenergic stimulation. We show that β₁ARs contribute to normal contractility at baseline, and it is the β₁AR that transduces the catecholamine signal to augment heart rate and contractility. In addition, it is the β₁AR that uniquely transmits the intracellular cascade that leads to the activation of CaMKII and CaMKII-dependent downstream signaling. Moreover, it appears that β₁ARs exert a predominantly adverse influence on postinfarction remodeling as shown by the worsening of cardiac function after myocardial injury, activation of myocardial CaMKII activity and increased apoptosis in the infarct border zone. In contrast, mice lacking β₁ARs (i.e., β₁AR KO and DKO mice) show a significantly attenuated decline in cardiac function post-MI that is not associated with enhanced CaMKII activity or increased in border zone apoptosis.

Several studies have reported cardiac responses after catecholamine stimulation as measured by changes in heart rate, blood pressure, and LV dP/dt in βAR KO mice (13, 26, 42–44). Although LV dP/dt_max provides useful information on relative changes in contractile function, it has limited sensitivity when groups of different animals are compared and therefore analysis of P-V relations is considered the most rigorous approach in the evaluation of cardiac contractility (21, 25, 36). Using this more sophisticated hemodynamic evaluation we clearly show the importance of β₁ARs in augmenting contractility in response to catecholamine infusion.

Our data suggest that one of the potential mechanisms contributing to the β₁AR-mediated deterioration in post-MI cardiac function could be due to enhanced activation of CaMKII. Recent studies suggest that activation of CaMKII may promote adverse cardiac remodeling, since overexpression of CaMKII can induce cardiomyopathy and inhibition of CaMKII by transgenic overexpression of an inhibitory peptide blocks cardiac deterioration post-MI (52, 53). At least two mechanisms have been recently identified that could potentially contribute to this pathological role of CaMKII following myocardial injury: 1) phosphorylation of the class IIa histone deacetylase (HDAC) 4 (6), and 2) phosphorylation of the cardiac ryanodine receptor (RYR2) (2). In the heart, is has recently been shown that CaMKII signals specifically to HDAC4 to promote its phosphorylation, nuclear export, and derepression of HDAC target genes that are involved in cardiac hypertrophic response (53). The action of CaMKII to phosphorylate the RYR2 and enhance SR diastolic Ca²⁺ leak (2, 22) and to phosphorylate Na⁺ channels (46) likely contributes to the contractile dysfunction and arrhythmogenesis in the failing heart (4, 32, 33). Indeed, KO of CaMKIIδ in mice results in attenuated LV chamber enlargement and cardiac dysfunction after pressure overload (31). These morphological remodeling changes were associated with decreased Ca²⁺ spark frequency and RYR2 Ca²⁺ leak in the CaMKII KO mice (22).

Previous studies have shown relationship between the level of CaMKII activity and apoptosis (47), which is consistent with our present data showing increased apoptosis in the hearts with enhanced CaMKII activity. Furthermore, β₁AR-mediated development of apoptosis can be prevented with transgenic overexpression of a CaMKII inhibitor (50). When these mice are crossed with PLB-deficient mice, which have increased susceptibility to catecholamine-induced apoptosis, inhibition of CaMKII abolished the resistance to apoptosis (50). Apoptosis has been implicated in cardiac ischemic injury and is likely involved in the transition from cardiac hypertrophy to decompensated heart failure, particularly after MI-induced LV dysfunction (45). Moreover, apoptosis of cardiac myocytes appear to peak during the first several days following MI in the ischemic area with little apoptosis found in remote regions of the myocardium from the injury (8, 28). These findings are consistent with our study showing increased apoptosis in the ischemic border zones and not in the remote areas of the myocardium. Importantly, we show that mice lacking β₁AR reveals significantly less apoptosis, suggesting that β₁AR activation promotes robust apoptosis. Similar data have been reported in cultured adult myocytes (14, 56). Although we demonstrate significant less apoptosis in the border zone of the β₁AR KO and DKO mice, our study was not designed to test whether this would translate into reduced infarct size since infract size was matched across the four groups. Indeed, a number of studies have shown beneficial effects of reducing cardiomyocyte apoptosis on ventricular function in a variety of heart failure models (17).

Prior studies have suggested that stimulation of β₂ARs may provide a protective mechanism in the heart by activating PI3K/Akt signaling (56), in part through their ability to switch coupling from G_s to G_i (16). It has been proposed that βAR-G_i cardioprotective signaling protects against myocyte apoptosis, while simultaneously attenuating detrimental β₂AR -G_s inotropism (54). However, since the level of apoptosis and cardiac dysfunction was similar in both β₁ARKO and DKO mice, our data support an alternate concept for the in vivo heart during pathological stress. That is, in response to myocardial injury β₂AR provide little in the way of cardioprotection, whereas β₁AR markedly stimulate detrimental signaling pathways.

Recently, Palazzesi et al. (37) assessed the role of βARs in the hypertrophic response to pressure overload. Consistent with our data, basal contractility in hearts of double βAR KO mice was similar to WT mice (37). Although response to catecholamine infusion was not performed, they showed that the hypertrophic response was not attenuated in banded double βAR KO mice, consistent with the concept that G_q-coupled receptors are the primary G protein-coupled receptors involved in the development of cardiac hypertrophy following pressure overload (3).

β₁AR account for majority of the βARs on the cardiac myocytes and are significantly downregulated in chronic human heart failure (41). Only hearts from WT and β₂AR KO mice showed marked reduction in plasma membrane receptor levels after MI consistent with β₁AR downregulation. In contrast, we did not observe any change in the receptor levels in the β₁ARKO mice following MI, suggesting little downregulation of β₂AR as seen in humans. Since we did not measure the level of β₃ARs in the βAR KO mice, we cannot exclude a possible contribution of β₃ARs to the attenuated adverse remodeling seen in the DKO mice. However, in transgenic mice with cardiac overexpression of human β₃ARs, selective β₃AR stimulation resulted in an increase in contractility that was entirely G_s dependent (27), suggesting that signaling via the β₃AR is unlikely to account for the observed phenotype. Finally, our data are consistent with a number of studies using β1 selective and nonselective β-antagonists and KO mice with regard to their effect on LV remodeling following pressure overload (26, 29, 38). However, in this study we demonstrate the importance of CaMKII activation as a potential mechanism for the adverse postinfarct remodeling and further show the relative contribution of both βAR subtypes in this response.

In summary, we demonstrate that cardiac contractility and CaMKII is mediated almost exclusively by β₁AR under basal and catecholamine-stimulated conditions. Moreover, we show that β₁AR signaling promotes detrimental cardiac remodeling following MI. Although multiple mechanisms are likely to be involved in the development of heart failure, our study supports the concept that β₁ARs activate CaMKII leading to enhanced apoptosis, both of which can potentially contribute to cardiac dysfunction. Contrary to the previous reports of the protective effects of β₂AR signaling, our findings from the β₁ARKO and DKO suggest that β₂AR do not actively contribute to the antiapoptotic components during the development of heart failure following MI. Taken together, these findings show that the β₁AR has a predominant influence on cardiac function in response to adrenergic stimulation and the pathological remodeling that follows MI.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-61558 (H. A. Rockman).