Reversal of Angiotensin-(1–12)-Caused Positive Modulation on Left Ventricular Contractile Performance in Heart Failure: Assessment by Pressure-Volume Analysis

Abstract

Background.

Angiotensin-(1–12) [Ang-(1–12)] is a renin-independent precursor for direct angiotensin-II production by chymase. Substantial evidence suggests that heart failure (HF) may alter cardiac Ang-(1–12) expression and activity; this novel Ang-(1–12)/chymase axis may be the main source for angiotensin-II deleterious actions in HF. We hypothesized that HF alters cardiac response to Ang-(1–12). Its stimulation may produce cardiac negative modulation and exacerbate left ventricle (LV) systolic and diastolic dysfunction.

Methods and Results.

We assessed the effects of Ang-(1–12) (2 nmol/kg/min, iv, 10 min) on LV contractility, LV diastolic filling, LV-arterial coupling (AVC) in 16 SD male rats with HF-induced by isoproterenol (3 mo after 170 mg/kg sq. for 2 consecutive days) and 10 age-matched male controls. In normal controls, versus baseline, Ang-(1–12) increased LV end-systolic pressure, without altering heart rate, arterial elastance (E_A), LV end-diastolic pressure (P_ED), the time constant (τ) and ejection fraction (EF). Ang-(1–12) significantly increased the slopes (E_ES and M_SW) of pressure (P)-volume (V) relations, indicating increased LV contractility. AVC (quantified as E_ES/E_A) improved. In contrast, in HF, versus HF baseline, Ang-(1–12) produced a similar increase in P_ES, but significantly increased τ, E_A, and P_ED. The early diastolic portion of LV P-V loop was shifted upward with reduced in EF. Moreover, Ang-(1–12) significantly decreased E_ES and M_SW, demonstrating decreased LV contractility. AVC was decreased by 43%.

Conclusions.

In both normal and HF rats, Ang-(1–12) causes similar vasoconstriction. In normal, Ang-(1–12) increases LV contractile function. In HF, Ang-(1–12) has adverse effects and depresses LV systolic and diastolic functional performance.

1. Introduction

Despite advances in its treatment, the prevalence of heart failure (HF) is increasingly high and associated with a poor prognosis [1]. Activation of the renin–angiotensin system (RAS) is pivotal for the development and progression of HF [2]. Angiotensin-(1–12) [Ang-(1–12)], a newly identified component of RAS, is a renin-independent precursor for direct angiotensin II (Ang II) formation by chymase in rodent and human heart tissue [3]. Recent studies demonstrate that this non-canonical chymase/Ang-(1–12) pathway may play an important role in modulating cardiac function bypassing cardioprotective actions of angiotensin-converting enzyme inhibitors (ACEI) and Ang II receptor blockers (ARBs) [4–6]. This interpretation rests on a documented limited efficacy of RAS blockade (ACEI or ARB) to reach the intracellular sites at which Ang II influences myocardial contractility and trophic actions. In keeping with this hypothesis, major clinical trials document a residual risk of clinical events of a magnitude 3 or more folds higher than the benefit [6–8]. To date, few have recognized the importance of Ang-(1–12) as a source for intracellular cardiac Ang II actions. Research from this laboratory suggests that human left heart diseases [9] and the presence of resistant atrial fibrillation is associated with higher cardiac Ang-(1–12) and chymase expression [10]. However, the changes in functional responses to Ang-(1–12) in normal and HF remain to be fully characterized. We have previously reported blunting of the positive inotropic effect of Ang-(1–12) on myocyte contractility and [Ca²⁺]_I transient in HF [11]. On the other hand, the direct cardiac effects of Ang-(1–12) on left ventricular (LV) contractility independent of alterations in loading conditions in normal vs. HF remain to be determined.

The aim of the present study was to examine the in vivo Ang-(1–12)-induced alternations in global cardiac function in HF vs normal. We tested the hypotheses that Ang-(1–12) may produce cardiac negative modulation and exacerbate LV systolic and diastolic dysfunction and contribute to the functional impairment of HF. With this in mind, we document the effect of Ang-(1–12) superfusion on LV systolic and diastolic performance in normal rats and in rats with isoproterenol-induced HF. This experimental HF model mimics many of the structural, functional, and hormonal changes of clinical HF [12, 13].

2. Methods

2.1. Animal Model

This study was approved by the Wake Forest School of Medicine Animal Care and Use Committee and conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication 8th edition, updated 2011). Thirty-six male Sprague–Dawley (SD) rats weighing 280–300 g (Charles River Laboratories International, Inc.) were used for this experiment. Rats were randomly divided into control (n=10) and HF groups (n=16). HF was induced by two subcutaneous injections of isoproterenol (ISO) spaced 24 hours apart at a dose of 170 mg/kg [13–16]. This procedure was successful in all but two of the injected rats. Rats injected with ISO were studied 3 months after the initial injection. All animals were maintained in the same environment, including temperature and humidity, and had free access to food and water.

2.2. Experimental protocol

We measured LV systolic and diastolic functional responses before and after Ang-(1–12) administration in both normal and HF rats.

2.3. Hemodynamic and Pressure-Volume Relation Measurements

Hemodynamic measurements were performed in normal and HF animals. Briefly, the rats were anesthetized with a combination of intraperitoneal ketamine (50 mg/kg) and xylazine (10 mg/kg). A heating pad was placed underneath each rat and the core temperature was maintained at 37°C. Animals were intubated and ventilated with a positive-pressure respirator (Model RSP1002, Kent Scientific Corp, Litchfield, CT) with oxygen-enriched room air with isoflurane (0.5–2%) to maintain anesthesia and arterial oxygen tension. The effective levels of anesthesia were checked every 10 to 15 minutes and were obtained and maintained by observing reactions to physical stimulation such as toe pinch, as well as monitoring the pattern and rate of respiration and changes in heart rate and blood pressure. A polyethylene catheter was inserted into the left external jugular vein for drug administration. As we described previously [17], after adequate calibration, using our well-established technique, a 2-F microtip P-V catheter (SPR-869, Millar Instruments, Houston, TX) was inserted into the right carotid artery and advanced into the LV under pressure control. After stabilization for 10 min, signals were continuously recorded at a sampling rate of 500 samples/s using a P-V conductance system (MPCU-200, Millar Instruments, Houston, TX) with BioBench software (National Instruments, Inc). Variables were recorded under steady-state conditions and during transient inferior vena caval occlusion (VCO). After baseline data collections, Ang-(1–12) (2 nmol/kg/min, iv) was infused continuously through a catheter which increased P_ES by ≈40 mm Hg. The dosing protocol of Ang-(1–12) used in this current study was chosen given that this dose was associated with robust increases in plasma Ang-(1–12) concentrations [18]. Steady-state and VCO data were continuously acquired immediately and for 15 minutes following Ang-(1–12) administration. This sequence was completed by repeating the study in the presence of the Ang II AT₁ receptor (AT₁-R) blocker losartan (LOS) (1 mg/kg plus 50 ng/kg/min, IV). We confirmed that this dosage of LOS produced adequate AT₁-R blockade by evaluating the response of arterial blood pressure to an infusion of Ang II [19, 20]. After hemodynamic study or blood collections, animals were given a lethal injection of sodium pentobarbital (100 mg/kg, iv) through the left external jugular vein catheter. The hearts were removed quickly.

As previously described by our laboratory [17] and others [21], with the use of a special P-V analysis program (PVAN, Millar Instruments, Houston, TX), standard steady-state hemodynamic parameters, such as heart rate, LV pressures (P) and the time constant of LV relaxation (τ) and LV volume (V), were measured. Stroke volume (SV) and cardiac output (CO) were calculated and corrected according to in vitro and in vivo volume calibrations using PVAN software [17] and others [21]. LV P-V relations and its slopes were derived. Effective arterial elastance (E_A) was calculated as the ratio of end-systolic pressure (P_ES) and SV, and LV-arterial coupling was quantitated as the ratio of the slope of linear P_ES-V_ES relation (E_ES) to E_A. [16, 21–25]

2.4. Statistical Analysis

All data are presented as mean ± SD. Multiple comparisons were performed using analysis of variance. When a significant overall effect was present, intergroup comparisons were performed using a Bonferroni correction for multiple comparisons. Two-tailed unpaired Student’s t-tests were used to evaluate mean differences in hemodynamic parameters between groups. Significance was established as p<0.05.

3. Results

3.1. Verification of Experimental HF

The rat model of ISO-induced HF has been validated by many investigators, including our laboratory [11,14,15]. Pathologic cardiac changes in ISO-treated rats resemble those of myocardial infarction (including LV structural remodeling, myocardial necrosis, eccentric hypertrophy with reduced contractile functional performance, and decreased β-adrenergic reserve) [12, 13, 15, 26]. Our current findings are in good agreement with the past reports from other groups. In the present study, 16 rats in the HF group survived after ISO injection. All had clear evidence of HF (anorexia and pulmonary congestion). In line with our previous work and that of others [13, 15], the total infarction area in ISO-injected rats averaged about 35 ± 4%. Diffuse subendocardial necrosis and pronounced LV enlargement with increased LV volume were also observed. Although body weights were similar in normal and HF rats (541 ± 28 vs. 535 ± 26 g, p=NS), cardiac weight (2.31 ± 0.34 vs. 1.64 ±0.29 g, p<0.05), ratio of heart-to-body weight (4.26 ± 0.72 vs. 3.18 ± 0.41 g/kg, p<0.01), and the wet lung-to-body weight ratio (4.25 ± 0.89 vs. 3.16 ± 0.74 g/kg, p<0.01) were all significantly increased in HF rats. These findings documented the existence of established HF in this model.

As presented in Table 1, compared to normal rats, HF rats showed significant increases in LV end-diastolic volume (394.1 vs. 320.9 μl, p<0.01) and LV end-systolic volume (272.6 vs. 151.9 μl, p<0.01) with reduced stroke volume (SV) (121.5 vs. 169.0 μl, p<0.01) and ejection fraction (31 vs. 60 %, p<0.01). The time constant of LV relaxation (τ, 16.0 vs. 9.7 msec) was increased by 65% (p<0.01). As shown in Table 2, LV contractility decreased >30% as measured by the slope of linear P_ES-V_ES relation (E_ES) (0.67 vs. 1.00 mm Hg/μl, p<0.01) and the slope of stroke work-end-diastolic volume (V_ED) relation (M_SW) (79.6 vs. 101.8 mm Hg, p<0.01).

Table 1.

Effects of Ang-(1–12) on Steady-State Hemodynamics in Normal and after HF

	Control (n=10)		HF (n=16)
	Baseline	Ang-(1–12)	Baseline	Ang-(1–12)
Heart rate (beats/min)	254±36	255±35	250 ± 24	248±24
Stroke Volume (μl)	169.0±17.6	228.1±16.0†	121.5 ± 21.9 *	93.2±21.6†‡
Ejection Fraction (%)	60±3	65±3†	31 ± 5*	23±5†‡
LV end-diastolic pressure (mm Hg)	5.5±1.1	6.0±0.7	14.2 ± 1.6 *	18.3±1.2†‡
LV end-systolic pressure (mm Hg)	101±6	144±6†	107 ± 5	145±13†
LV end-diastolic volume (μl)	320.9±23.8	335.1±21.4	394.1 ± 17.5*	402.0±19.4†‡
LV end-systolic volume (μl)	151.9±28.5	106.9±19.8†	272.6 ± 16.6 *	308.9±12.6†‡
Maximum dV/dt (μl /sec)	6,954±864	6,929±730	4,683 ± 335 *	3,547±268†‡
Time constant of relaxation (msec)	9.7±0.6	9.6±0.7	16.0 ± 2.4 *	23.0±2.7†‡
EA (mm Hg/μl)	0.60±0.06	0.63±0.04	0.91 ± 0.15 *	1.63±0.34†‡

Values are means ± SD. n= number of rats.

LV, left ventricular; maximum dV/dt, the peak rate of mitral flow; E_A, arterial elastance.

^*P <0.05, HF baseline vs. control baseline.

^†P <0.05, Ang-(1–12) vs. corresponding baseline value.

^‡p<0.05, Ang-(1–12)-induced changes after HF vs. Ang-(1–12)-induced changes in normal.

Table 2.

Effects of Ang-(1–12) on LV Pressure-Volume Relations and Arterial Coupling in Normal and after HF

	Control (n=10)		HF (n=16)
	Baseline	Ang-(1–12)	Baseline	Ang-(1–12)
EES (mm Hg/μl)	1.00 ± 0.12	1.30±0.14†	0.67 ± 0.05*	0.54±0.05†‡
MSW (mm Hg)	101.8 ± 8.9	121.9±9.2†	79.6 ± 5.2*	60.6±4.7†‡
EES/EA	1.67 ± 0.18	2.06±0.20†	0.76 ± 0.16*	0.35±0.04†‡

Values are means ± SD. n= number of rats.

E_ES, the slope of linear P_ES-V_ES relation; M_SW, the slope of stroke work–V_ED relation.

^*P <0.05, HF baseline vs. control baseline.

^†P <0.05, Ang-(1–12) vs. corresponding baseline value.

^‡p<0.05, Ang-(1–12)-induced changes after HF vs. Ang-(1–12)-induced changes in normal.

3.2. Effects of Ang-(1–12) on hemodynamics and LV functional performance

The steady-state hemodynamic measurements at baseline and after Ang-(1–12) in normal control and in HF are summarized in Table 1. LV pressure-volume analysis in normal control and in HF before and after Ang-(1–12) are presented in Table 2. Figure 1 displays the examples of the effects of Ang-(1–12) on steady-state LV P-V loops (A) and LV P_ES-V_ES in normal control and after HF.

Figure 1.

Examples of the effects of Ang-(1–12) on steady-state LV P-V loops (A) and LV P_ES-V_ES relations (B) from one normal control rat and one rat after HF.

A. In normal rat, Ang-(1–12) administration caused no marked changes on the P-V loops. In contrast, after HF, Ang-(1–12) produced a rightward and upward shift of the P-V loop with marked increases in LV P_min and P_ED, indicating impaired LV diastolic performance.

B. In normal, Ang-(1–12) caused a leftward shift of P_ES-V_ES relations with increased slopes, indicating increased LV contractility. After HF, Ang-(1–12) caused a rightward shift of P_ES-V_ES relations with decreased slopes, indicating decreased LV contractility.

Normal Control:

Compared with normal control at baseline, Ang-(1–12) produced no change in heart rate, arterial elastance (E_A) [Ang-(1–12): 0.63 vs. 0.60 mm Hg/μl, p = >0.05], τ (9.7 vs. 9.6 msec, p = NS, >0.05), end-diastolic pressure (P_ED) (6.0 vs. 5.5 mm Hg, p = NS, >0.05) were relatively unchanged, but significantly increased end-systolic pressure (P_ES) [Ang-(1–12): 143.5 vs. baseline: 100.5 mm Hg], stroke volume (228.2 vs. 169.0 μl) and EF (65.3 vs. 60.3%) (p <0.01) (Table 1 and Figure 1A). In normal rats, Ang-(1–12) caused significantly increased the slopes of Pressure (P)-Volume (V) relations of E_ES (1.3 vs 1.0 mm Hg/μl) and M_SW (121.9 vs 101.8 mm Hg), indicating increased LV contractility. LV-arterial coupling (AVC), quantified as E_ES/E_A, was significantly improved. This indicated that in normal rats, Ang-(1–12) displayed a positive inotropic effect on LV contractile performance and did not markedly alter LV diastolic filling dynamics (Table 2 and Figure 1B).

Heart Failure:

In HF rats, Ang-(1–12) produced no marked change in heart rate with a similar increase in P_ES (145.4 vs. 107.2 mm Hg, p<0.01). Compared with HF baselines, after Ang-(1–12), the τ (23.3 vs 16.3 msec), E_A (1.6 vs 0.9 mm Hg/μl, p<0.01) and P_ED (18.3 vs 14.1 mm Hg, p<0.01) were all significantly increased. The early diastolic portion of LV P-V loop was also shifted upwardly with reduced EF (23 vs 31%, p<0.01) and SV (93.2 vs 121.5 μl, p<0.01) (Table 1). Furthermore, compared with HF at baseline, Ang-(1–12) caused rightward shifts and significantly decreased the slopes of P-V relations of E_ES (0.54 vs 0.67 mm Hg/μl) and M_SW (60.6 vs 79.6 mm Hg), demonstrating decreased LV contractility (Table 2 and Figure 1B). LV-arterial coupling (E_ES/E_A) was also decreased about 50% (0.4 vs 0.8). This indicates that in HF, Ang-(1–12) produced a direct negative inotropic effect on LV contractile performance. In addition, compared to HF baseline, Ang-(1–12) caused rightward and upward shifts of the early diastolic portion of the LV P-V loop in HF rats with increased early diastolic LV pressure and τ (23.0 vs. 16.0 msec, p<0.01). The peak rate of mitral flow, Maximum dV/dt significantly reduced (3,547 vs. 4,683 μl/s, p<0.01). These changes were completely reversed by LOS, a selective AT₁-R antagonist.

4. Discussion

Our study demonstrates, for the first time, the effect of exogenous Ang-(1–12) on LV systolic and diastolic performance in normal and HF. The major findings of this investigation are: (1) In normal rats, Ang-(1–12) infusion produced positive inotropic effects in the LV with significantly improved LV filling and LV-arterial coupling; and (2) In HF, exogenous Ang-(1–12) depresses LV contraction and relaxation, accompanied with significantly impaired LV filling and LV-arterial coupling. This suggests that Ang-(1–12) may contribute to the impairment of both systolic and diastolic functional performance in HF. Thus, renin-angiotensin system activation with HF may have adverse functional consequences that are due to an altered response to Ang-(1–12) in HF. Our findings not only provide significant and timely knowledge of how Ang-(1–12) may act in the heart as an autocrine/paracrine hormone to affect cardiac function, but also for potentially uncovering new approaches for improving the efficacy of current therapeutic regimens. This investigation suggests that drug-treatment approaches that inhibit Ang-(1–12) and its pathway within currently existing HF therapies may provide added benefit to patients with HF.

Ang-(1–12) is a potent vasoconstrictor and acts as an endogenous substrate for Ang II production [3]. The dose of Ang-(1–12) used in this study was associated with robust increases in plasma Ang-(1–12) concentrations and significant changes in arterial pressure [18]. To avoid the potentially confounding influences of these changes in loading conditions on conventional measures of LV performance, we evaluated LV contractile performance in the variably loaded pressure-volume loops. We found that in normal rats, Ang-(1–12) had direct positive inotropic effects with the increased E_ES and M_SW. It appears that the direct positive inotropic effect of Ang-(1–12) overcomes the effect of arterial vasoconstriction. In HF rats, Ang-(1–12) produced a similar increase in LV end-systolic pressure without altering the heart rate. In contrast, compared with normal, Ang-(1–12) produced a significant depression in LV contractile performance as indicated by the rightward shift of the LV P-V relations and decreased slopes and M_SW. Thus, the negative inotropic effects of Ang-(1–12) in HF rats were not dependent on changes in loading conditions and heart rate.

What is the mechanism of Ang-(1–12)’s depression of LV contraction and relaxation in HF? Ang-(1–12) serves as a functional substrate for Ang II formation, as the peptide showed vasoconstrictor actions in isolated rat aortic strips. Moreover potent pressor responses produced by systemic Ang-(1–12) administration were blocked by either captopril or candesartan [3]. In HF, the heart is more sensitive to increased afterload, thus it is possible that the decreased LV systolic performance and slowed relaxation we observed with Ang-(1–12) in HF was due to an Ang II-induced increase in afterload. However, consistent with our past report [27], the effects of Ang-(1–12) was not completely reversed when P_ES was returned to baseline with nitroprusside, indicating that Ang-(1–12) has a direct role impairing systolic and diastolic performance in HF (data not shown). This direct cardiac action of Ang-(1–12) may account for the findings of contrasting changes of Ang-(1–12) effects in the normal vs. HF LV. This is consistent with our previous study showing that superfusion of Ang-(1–12) to isolated myocytes causes direct decreases in myocyte contractility, and relaxation rate, and impairs [Ca²⁺]i regulation [11]. Since these effects are present in the isolated myocytes, they are not due to potentially confounding effects of extra-cardiac factors that may influence contractility. Ang-(1–12) can interact with membrane-bound chymase when it reaches cardiac tissue, leading to Ang II production [5]. In our study, this Ang II-mediated effect can be blocked by losartan. The Ang II generated either from ACE in the systemic circulation or from alternate chymase processing pathway may interact with Ang II AT₁ receptor (AT₁-R) via autocrine/paracrine mechanism [4, 28–30]. It’s been shown that Ang II and Ang-(1–12)-mediated contractile action couples to Gs-proteins in normal myocytes, which increases cardiac contractility [11, 31, 32]. In HF, Ang II AT₁-R and Ang-(1–12)-mediated contractile action may couple to an inhibitory G protein, Gi [11]. Thus the cAMP-dependent intracellular signal transduction system is disrupted, which may exacerbate the dysfunctional Ca²⁺ homeostasis, resulting in an altered inotropic effect on myocardial contraction and relaxation as we observed after HF [33, 34].

Despite evidence for the involvement of Ang II in the pathogenesis of CV disease, the long-term use of RAS inhibitors in the treatment of patients with CV disease demonstrated an imbalance between patients that significantly benefit from these therapeutic agents and those that remain at risk for heart disease progression [35]. One explanation for this is that ACE inhibitors or ARBs do not reach the intracellular compartment at which Ang II acts [36]. It’s been reported that Ang-(1–12) forms Ang II intracellularly and is regulated independently from the circulating RAS [5]. In normal human LV plasma membranes Ang-(1–12) is directly converted into Ang II by chymase [37]. In pathological states, Ang-(1–12) expression was increased and may contribute to intracrine Ang II formation and its long-term harmful effects [38, 39]. According to Komatsu et al, [40] subacute infusion of Ang-(1–12) exerts pressor effects accompanied with cardiac hypertrophy in an ACE and AT₁-R-dependent manner. The current findings of Ang-(1–12)-induced a direct depression of LV contractile and diastolic performance in HF are in line with our hypothesis that Ang-(1–12) acts as a non-canonical mechanism for intracellular Ang II action and the upregulation of Ang-(1–12) may play a major role in the adverse remodeling and functional decline of the failing heart. This knowledge may impart new therapeutic opportunities to prevent Ang II broad pathological functions in HF by preventing Ang-(1–12) from being processed into Ang II.

Moreover, we found that in HF, Ang-(1–12) also significantly impaired LV-arterial coupling. We showed previously that LV-arterial coupling, which regulates SV at a given preload and closely relates to LV mechanical efficiency, is a key determinant of optimal cardiovascular function [41]. In HF, E_ES is significantly decreased, but E_A is increased, reducing the E_ES/E_A ratio and resulting in less maximal stroke work (SW) [41, 42]. We showed that this impaired LV-arterial coupling was exacerbated after Ang II administration [43, 44]. In this study, in the normal controls, Ang-(1–12) administration did not significantly alter E_A, but significantly increased E_ES, so the E_ES/E_A ratio increased. However, in HF, Ang-(1–12) caused a significant increase in E_A and a decrease in E_ES, so LV-arterial coupling worsened (E_ES/E_A ratio). This new finding extends our understanding of the detrimental role of Ang-(1–12) in heart disease. In support for this argument, chymase inhibition, which may decrease Ang II production by Ang-(1–12), prevented cardiac remodeling and diastolic function improvement in HF [45], and improved myocardial performance and survival following myocardial infarction [46].

5. Study limitations

Our study has several limitations. Although pathologic changes in ISO-treated rats resemble those of myocardial infarction [12] and ISO induced HF mimics many structural, functional, and neurohormonal changes of clinical HF [12, 15], we cannot ascertain that these results would apply to clinical HF or HF from other causes, such as pure pressure overload cardiomyopathy or pure volume overload. Second, the Ang-(1–12) doses employed here were based on a concentration-response study in the past report [18]. It is not known whether lower doses of Ang-(1–12) may influence results. Third, the purpose of this study was to test the direct cardiac effect of Ang-(1–12) in HF, which is processed into Ang II either by ACE in the systemic circulation or chymase in the heart [5, 37]. Our study was not designed to answer the metabolic pathway by which Ang-(1–12) infusion resulted in Ang II formation. While future studies will address this question and more insights will be gained.

6. Conclusions

HF alters LV functional responses to Ang-(1–12). In normal, Ang-(1–12) increases LV contractile function. In HF, Ang-(1–12) has adverse effects and depresses LV systolic and diastolic functional performance. These effects are mediated by AT₁-R and may involve Ang II formation by chymase. This study uncovers a potential new molecular target for HF management.

Highlights:

• Ang-(1–12) augments LV contractile performance of the normal heart.

• In heart failure, Ang-(1–12) depresses LV systolic and diastolic functional performance.

• Ang-(1–12) inotropic responses on LV functional performance are mediated by the activation of Ang II AT₁ receptors.

• Cardiac chymase may account for Ang II generation from Ang-(1–12).