Restoration of Mechanical and Energetic Function in Failing Aortic-Banded Rat Hearts by Gene Transfer of Calcium Cycling Proteins

Abstract

The aim of this study was to examine whether short- and long-term gene transfer of Ca²⁺ handling proteins restore left ventricular (LV) mechanoenergetics in aortic banding-induced failing hearts. Aortic banded rats received recombinant adenoviruses carrying sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) (Banding+SERCA), parvalbumin (Banding+Parv) or β-galactosidase (Banding+βgal), or an adeno-associated virus carrying SERCA2a (Banding+AAV.SERCA) by a catheter-based technique. LV mechanoenergetic function was measured in cross-circulated hearts. “Banding”, “Banding+βgal” and “Banding+saline” groups showed lower end-systolic pressure at 0.1 ml intraballoon water (ESP_0.1), higher end-diastolic pressure at 0.1 ml intraballoon water (EDP_0.1) and slower LV relaxation rate, compared with “Normal” and “Sham”. However, “Banding+SERCA” and “Banding+Parv” showed high ESP_0.1, low EDP_0.1, and fast LV relaxation rate. In “Banding”, “Banding+βgal” and “Banding+saline”, slope of relation between cardiac oxygen consumption and systolic pressure-volume area, O₂ cost of total mechanical energy, was twice higher than normal value, whereas slope in “Baning+SERCA” and “Banding+Parv” was similar to normal value. Furthermore, O₂ cost of LV contractility in the 3 control banding groups was ∼3 times higher than normal value, whereas O₂ cost of contractility in “Banding+SERCA”, “Banding+AAV.SERCA” and “Banding+Parv” was as low as normal value. Thus, high O₂ osts of total mechanical energy and of LV contractility in failing hearts indicate energy wasting both in chemomechanical energy transduction and in calcium handling. Improved calcium handling by both short- and long-term overexpression of SERCA2a and parvalbumin transforms the inefficient energy utilization into a more efficient state. Therefore enhancement of calcium handling either by resequestration into the SR or by intracellular buffering improves not only mechanical but energetic function in failing hearts.

1. Introduction

Calcium handling is central to the process of excitation-contraction (E-C) coupling. Sarcoplasmic reticulum (SR), which releases Ca²⁺ during contraction by a trigger of Ca²⁺ entry and takes it up during relaxation by SR-ATPase (SERCA2a) pump, plays a major role in controlling the synchronized Ca²⁺ handling in myocardial cells [1]. Removal of Ca²⁺ from the cytoplasm is governed mainly by SERCA2a, of which the activity is regulated by phospholamban, and to a lesser extent by Na⁺-Ca²⁺ exchanger (NaCaX). SERCA2a protein/mRNA expression and activity were decreased in human heart failure and in animal models of heart failure [2,3]. This SERCA2a reduction results in abnormal Ca²⁺ handling, which causes a prolongation of Ca²⁺ transient, an increase in diastolic intracellular Ca²⁺, a decrease in systolic intracellular Ca²⁺ and a reduced SR Ca²⁺ content. Therefore, abnormal Ca²⁺ handling by the SERCA2a reduction contributes to the systolic and diastolic dysfunction in failing hearts [4,5].

Our group [6-11] has previously shown that overexpression of SERCA2a, ablation of phospholamban, or overexpression of parvalbumin by adenoviral gene transfer modify intracellular Ca²⁺ handling and modulate physiological mechanical performance in normal, senescent, aortic banding-induced failing and ischemia/reperfusion-injured rat whole hearts. In addition, global cardiac gene transfer of SERCA2a improved survival and energetic state shown as phosphocreatine/ATP ratio in failing rat hearts induced by aortic banding [9], and reduced ventricular arrhythmias in the model rat of ischemia followed by reperfusion [10]. More recently, SERCA2a overexpression improved the energy utilization in special reference to myocardial oxygen consumption in diabetic failing hearts [12].

The aim of this study was to examine whether restoring calcium cycling by either re-sequestering the elevated intracellular calcium back into the SR (through SERCA2a gene transfer) or by buffering the elevated intracellular calcium (through parvalbumin gene transfer) would improve the left ventricular (LV) mechanical and energetic functions in the aortic banded rats, especially in terms of oxygen cost of LV contractility.

2. Materials and methods

2.1. Recombinant adenoviral vectors

Recombinant adenoviral vectors were used with cytomegalovirus-driven expression cassettes for SERCA2a, parvalbumin, or β-galactosidase with a second cassette in each adenovirus containing green fluorescent protein substituted for E1 by means of homologous recombination. Adenoviral SERCA2a (Ad.SERCA), adenoviral parvalbumin (Ad.Parv) and adenoviral β-galactosidase (Ad.βgal) had concentrations of 9 X 10¹⁰ pfu/ml, 10 X 10¹⁰ pfu/ml and 8 X 10¹⁰ pfu/ml, respectively, with a particle/pfu ratio of 10:1. Wild-type adenovirus contamination was excluded by the absence of PCR-detectable E1 sequences.

2.2. Recombinant adeno-associated virus (AAV) vectors

The AAV1 vector used in this study was manufactured using standard calcium phosphate transfection methods in 293 cells. Three plasmids were used, one containing helper functions from adenovirus, one containing the AAV rep2 and cap1 genes, and the third containing the vector genome. Vector production, harvest, purification and testing were as previously described [13].

2.3. Animals

All animal experiments in this study were performed with the approval of the Animal Care Committee of Massachusetts General Hospital and in accordance with the National Institute of Health′s Guide for the Care and Use of Laboratory Animals. Five-week-old male Wistar rats (Charles River, Mass; body weight (BW), 100 to 120 g) were anesthetized with intraperitoneal (i.p.) Ketamine-Xyla-Jet mixture and placed on a ventilator. An incision on the right side of chest was made, exposing the aortic root, and a tanatalum clip with an internal diameter of 0.58 mm (Weck Hemoclip) was placed on the ascending aorta. Animals in the sham group underwent a similar procedure without insertion of a clip. Twenty-four to twenty-six weeks later, about 90 % of the animals thus operated survived (aortic banding, 89%; sham-operation, 93%), and were randomized into 8 groups:

1. six normal control rats (Normal);

2. six uninfected sham-operated rats (Sham);

3. six uninfected aortic banded rats (Banding);

4. five aortic banded rats infected with Ad.βgal (Banding+βgal)

5. five aortic banded rats injected with saline (Banding+saline);

6. six aortic banded rats infected with Ad.SERCA (Banding+SERCA);

7. five aortic banded rats infected with Ad.Parv (Banding+Parv);

8. three aortic banded rats infected with AAV1 carrying SERCA2a (Banding+ AAV.SERCA).

2.4. Cardiac gene delivery

The in vivo cardiac gene transfer has been described previously in detail by our group [14]. Briefly, after the rats were anesthetized and a thoracotomy performed, a 22-guage catheter containing 200 μl of adenovirus and 50 μl adenosine (3 mg/ml) was advanced from the apex of the left ventricle to the aortic root. The aorta and main pulmonary artery were clamped for 30 seconds distal to the site of the catheter and the solution injected, then the chest was closed, and the animals were allowed to recover. In the group (4) - (8), 60-67% of the rats thus operated survived until LV mechanical and energetic studies (Banding+βgal, 60%; Banding+saline, 63%; Banding+SERCA, 62%; Banding+Parv, 64%; Banding+AAV.SERCA; 67%). The rats underwent LV mechanical and energetic studies 2-3 days after the injection of the adenovirus or saline and 45 days after the AAV injection.

2.5. LV mechanical and energetic studies

Surgical preparations

The LV mechanical and energetic studies were performed on the excised, cross-circulated rat heart preparations. The surgical preparations have been described previously in detail [15]. Briefly, in each experiment, 2 male 500 to 650g Wistar rats (blood supplier and metabolic supporter) and 1 heart donor rat were anesthetized with pentobarbital sodium (50 mg/kg, ip) and intubated and heparinized (1000 U, iv). In the beating heart, cross-circulated and maintained at 37°C, a thin latex balloon, which was connected to a pressure transducer for measuring LV pressure (LVP), was inserted into the LV. Systolic unstressed volume (V₀=0.08 ml for normal or sham-operated hearts and V₀=0.06 ml for aortic banded hearts) should be determined as the volume at which peak isovolumic pressure was zero. Heart rate was maintained constant at 300 beats/min by right atrium electrical pacing. Systemic arterial blood pressure of the support rat served as coronary perfusion pressure (diastolic-systolic, 80-120 mmHg; mean, about 90 mmHg). Arterial pH, Po₂, and Pco₂ of the support rat and perfused blood were maintained within their physiological ranges by supplemental oxygen and sodium bicarbonate throughout the experiment.

Calculation for oxygen consumption

Cardiac VO₂ was obtained as the product of coronary flow and arteriovenous O₂ content difference. The right ventricular (RV) component of total VO₂, which is considered constant irrespective of LV volume, was calculated by multiplying biventricular VO₂ under LV volume-unloading with the weight ratio of RV/RV+LV. The RV VO₂ was subtracted from the total VO₂ to yield LV VO₂.

Experimental protocol

LVP, LV VO₂ and systolic pressure-volume area (PVA) data were obtained at five different LV volumes, changed with a step of 0.025 ml, without any inotropic interventions (control volume run). After control volume run, Ca²⁺ inotropism run was performed at a midrange LV volume (mLVV) by intracoronary infusion of 1% CaCl₂ solution. In some experiments, dobutamine (78 μM) inotropism run was also performed after Ca²⁺ inotropism run. The steady state of heart was reached 2-3 min after change of LV volume and 4 min after change of the infusion rate. Cardiac arrest was induced by intracoronary infusion of 1M KCl (12 ml/h) to obtain VO₂ for basal metabolism. In each steady state, the data sampling, performed at 500 Hz for 2 s, was repeated 3 times at intervals of 0.5 min.

2.6. Data analysis

PVA calculation

The best-fit end-systolic/diastolic pressure-volume relations (ESPVR/EDPVR) were obtained by fitting the data with the exponential functions [12]. PVA was defined as the pressure-volume area circumscribed by the curvilinear ESVPR, EDPVR and the systolic portion of the ventricular pressure-volume trajectory.

VO₂ for Ca²⁺ handling in E-C coupling during inotropic run

During Ca²⁺/dobutamine inotropic run at a mLVV, ESP-volume relations at different infusion rates were obtained as a best-fit exponential function curve. We calculated PVA at mLVV (PVA_mLVV) by integrating each ESPVR from V₀ up to mLVV. We then obtained the two composite VO₂-PVA_mLVV data points. Next, the lines including each VO₂-PVA_mLVV data point were drawn in parallel to control VO₂-PVA relation. VO₂-intercept value (PVA-independent VO₂) at each infusion rate was thus obtained. VO₂ for Ca²⁺ handling in E-C coupling was obtained by subtracting basal metabolic VO₂ per beat, measured in KCl-induced arrest hearts, from PVA-independent VO₂.

LV contractility

Equivalent maximal elastance (eEmax), an index for LV contractility, was obtained by calculating ESP-volume ratio of the specific virtual triangle, which is energetically equivalent to the real PVA_mLVV [16].

Oxygen cost of LV contractility

The oxygen cost of LV contractility was obtained as the slope of the linear relation between VO₂ for Ca²⁺ handling in E-C coupling and eEmax during Ca²⁺/dobutamine inotropism run. This slope is considered an index quantifying Ca²⁺ handling VO₂ per unit LV contractility change.

Logistic time constant

To evaluate the LV relaxation rate, we analyzed LV isovolumic relaxation pressure-time curves at mLVV by using the logistic time constant (T_L) derived from a logistic model.

2.7. Western blot for SERCA2a protein

Lysates from hearts were matched for protein concentration and then separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were incubated with SERCA2a antibodies (Affinity Bioreagents, CA) followed by detection with enhanced chemi-luminescence.

2.8. Statistics

Data are presented as mean ± SD. Multiple comparisons were performed by ANOVA with STATVIEW (Abacus Concepts, Barkeley, CA). Statistical significance was accepted at the level of P<0.05.

3. Results

3.1. Characterization of animals

There was no statistical difference in BW among the 7 groups (Table 1). LV/BW ratio and LV+RV/BW ratio, as well as LV weight and LV+RV weight, in 5 aortic banding groups were significantly higher than those of “Normal” and “Sham” groups, while RV/BW ratio, as well as RV weight, was not different among 7 groups (Table 1). Thus, the rats, which underwent an aortic banding for 24-26 weeks, had a significant increase in LV mass normalized to BW, and a cross-section of the aortic banded hearts showed apparent LV concentric hypertrophy (data not shown).

Table 1.

Morphometric analyses

Group	n	BW (g)	LV (g)	RV (g)	LV+RV (g)	LV/BW (X10-3)	RV/BW (X10-3)	LV+RV/BW (X10-3)
Normal	6	598 ± 34	1.014 ± 0.076	0.288 ± 0.022	1.301 ± 0.084	1.69 ± 0.08	0.48 ± 0.05	2.18 ± 0.11
Sham	6	627 ± 53	1.091 ± 0.115	0.297 ± 0.049	1.388 ± 0.159	1.74 ± 0.06	0.47 ± 0.05	2.21 ± 0.10
Banding	6	614 ± 53	1.351 ± 0.127 a,b	0.302 ± 0.025	1.653 ± 0.143 a	2.21 ± 0.24 a,b	0.50 ± 0.07	2.71 ± 0.30a,b
Banding + βgal	5	607 ± 65	1.312 ± 0.176 a,b	0.306 ± 0.042	1.618 ± 0.207 a	2.16 ± 0.14 a,b	0.51 ± 0.05	2.66 ± 0.16 a,b
Banding + saline	5	648 ± 59	1.488 ± 0.164 a,b	0.312 ± 0.02	11.799 ± 0.160 a,b	2.29 ± 0.12 a,b	0.49 ± 0.07	2.78 ± 0.13 a,b
Banding + SERCA	6	580 ± 46	1.317 ± 0.138 a,b	0.325 ± 0.067	1.642 ± 0.195 a	2.28 ± 0.28 a,b	0.57 ± 0.15	2.85 ± 0.43 a,b
Banding + Parv	5	554 ± 52	1.277 ± 0.115 a,b	0.288 ± 0.023	1.565 ± 0.135 a	2.32 ± 0.24 a,b	0.52 ± 0.05	2.84 ± 0.29 a,b

All data are shown as mean ± SD. BW, body weight; LV, left ventricle; RV, right ventricle; n, number of hearts.

^aP < 0.05 compared to “Normal” group.

^bP < 0.05 compared to “Sham” group.

3.2. LV mechanics

Figure 1A shows representative control ESVPRs and EDPVRs in “Sham”, “Banding” and “Banding+SERCA” hearts. Curvilinear ESPVRs and EDPVRs of “Banding+βgal” and “Banding+saline” hearts were similar to those of “Banding” hearts (data not shown). ESPVR of “Banding+SERCA” and “Banding+Parv” hearts was shifted upward compared with ESPVR of “Banding” hearts. Summarized data of LV mechanics are shown in Table 2. The volume intercepts of ESPVR and EDPVR, V₀ and V_u, in 5 aortic banding groups were significantly smaller than those of “Normal” and “Sham” groups, resulting from LV concentric hypertrophy. In “Banding” hearts, the end-systolic pressure observed at 0.1 ml of intraballoon water volume (ESP_0.1), was lower (130 ±27 mmHg), and end-diastolic pressure observed at 0.1 ml of intraballoon water volume (EDP_0.1), was higher (22.1 ±6.2 mmHg), compared with “Normal” hearts (ESP_0.1,188 ±15 mmHg; EDP_0.1, 9.8 ±4.0 mmHg). However, in “Banding+SERCA” group, like in “Banding+Parv” group, ESP_0.1 was increased over 200 mmHg and EDP_0.1 was decreased to 12.4 ±2.7 mmHg, although ESP_0.1 in “Banding+βgal” and “Banding+saline” groups was as low as that of “Banding” group. “Banding+βgal” group showed a higher EDP_0.1, but without significant difference, compared with “Banding+SERCA” group. Two of “Banding+βgal” hearts had lower EDP_0.1 (12.3 and 12.6 mmHg), shorter logistic time constant, T_L (13.0 and 14.2 msec) and higher eEmax at mLVV (3538 and 3005 mmHg·ml^-1·g), compared with the rest of “Banding+βgal” hearts. Thus, in the two hearts, the systolic/diastolic cardiac function was not much deteriorated. The systolic/diastolic function was dependent on the levels of SERCA2a protein expression or SERCA2a activity [17,18]. In this study, therefore, the degree of cardiac dysfunction appears somewhat different among the aortic-banded animals because of the different levels of down-regulated SERCA2a expression. In all groups, moreover, T_L was obtained from LV isovolumic relaxation pressure-time curves at mLVV. T_L in “Banding” group was significantly longer than that of “Normal” and “Sham” groups (p<0.05). In “Banding+SERCA” and “Banding+Parv” groups, however, T_L was decreased to T_L level observed in “Normal” and “Sham” groups, although T_L in “Banding+βgal” and “Banding+saline” groups remained as long as that of “Banding” group.

Fig. 1.

(A) End-systolic volume relation (closed symbols) and end-diastolic pressure-volume relation (open symbols) in “Sham” (■and □), “Banding” (▲ and △) and “Banding+SERCA” (● and ○) hearts. Both relations were obtained by control volume-loading runs where balloon water was changed from 0 to 0.1 ml by 0.025 ml. (B) Comparison of logistic time constants among 7 groups. Logistic time constants were obtained from a best-fit logistic curve of pressure-time curve during isovolumic relaxation at midrange LV volume (0.05 ml of balloon water volume). *P<0.05 compared to “Normal” and “Sham” groups. ^#P<0.05 compared to “Banding”, “Banding+βgal” and “Banding+saline” groups. n=6 in “Normal”, n=6 in “Sham”, n=6 in “Banding”, n=5 in “Banding+βgal”, n=5 in “Banding+saline”, n=6 in “Banding+SERCA”, n=5 in “Banding+Parv”. (C) Linear relations between myocardial oxygen consumption per beat (VO₂) and systolic pressure-volume area (PVA) in “Sham” (■), “Banding” (▲) and “Banding+SERCA” (●) hearts.

Table 2.

Variables of LV mechanics

	ESPVR					EDPVR
Group	n	A (mmHg)	B (1/ml)	V0 (ml/g)	ESP0.1 (mmHg)	A′ (mmHg)	B′ (1/ml)	Vu (ml/g)	EDP0.1 (mmHg)
Normal	6	221±20	13.2±5.4	0.079±0.007	188±15	0.35±0.40	28.1±11.2	0.079±0.007	9.8±4.0
Sham	6	191±20	45.9±21.6	0.074±0.008	188±15	1.44±1.07	28.0±6.5	0.074±0.008	13.4±4.7
Banding	6	177±45	25.3±17.4	0.045±0.005a,b	130±27a,b	0.93±0.35	43.4±5.5	0.045±0.005a,b	22.1±6.2a
Banding +βgal	5	132±24a,b	57.0±26.8a	0.047±0.007a,b	128±27a,b	2.12±2.79	34.0±12.6	0.047±0.007a,b	15.1±3.0
Banding + saline	5	155±22a	27.3±16.3	0.040±0.005a,b	118±19a,b	2.45±1.74	37.4±7.4	0.040±0.005a,b	24.8±9.8a,b
Banding + SERCA	6	206±46d	65.6±30.1a	0.046±0.005a,b	210±46c,d,e	2.45±3.56	33.6±12.2	0.046±0.005a,b	12.4±2.7c,e
Banding + Parv	5	238±39d,e	47.6±18.3	0.047±0.004a,b	232±31c,d,e	1.83±1.13	33.7±10.9	0.047±0.004a,b	16.7±3.8

All data are shown as mean ± SD. ESPVR, end-systolic pressure-volume relation; A and B are parameters in the equation ESP=A{1-exp[-B(V-V₀)]};

V₀, volume intercept of end-systolic pressure-volume relation;

ESP_0.1, end-systolic pressure observed at maximum left ventricular volume (0.1 ml of balloon water volume);

EDPVR, end-diastolic pressure-volume relation; A′ and B′ are parameters in the equation EDP=A′{exp[B′(V-V_u)]-1};

V_u, volume intercept of end-diastolic pressure-volume relation;

EDP_0.1, end-diastolic pressure observed at maximum left ventricular volume (0.1 ml of balloon water volume); n, number of hearts.

^aP < 0.05 compared to “Normal” group.

^bP < 0.05 compared to “Sham” group.

^cP < 0.05 compared to “Banding” group.

^dP < 0.05 compared to “Banding +βgal” group.

^eP < 0.05 compared to “Banding + saline” group.

3.3. LV energetics; VO₂-PVA relations

Figure 1C shows representative control VO₂-PVA relations in “Sham”, “Banding” and “Banding+SERCA” hearts. “Banding” heart showed a linear VO₂-PVA relation with steeper slope compared with “Sham” and “Banding+SERCA” hearts. Summarized data of LV energetics are shown in Table 3. There was no significant difference in the VO₂ intercept of VO₂ -PVA relation among 7 groups. On the other hand, the slope in “Banding” group was about twice as high as that of “Normal” and “Sham” groups. However, the slope in “Banding+SERCA” and “Banding+Parv” groups was similar to the slope found in “Normal” and “Sham” groups, although the slope in “Banding+βgal” and “Banding+saline” groups remained high. In five aortic banding groups, moreover, the minute VO₂ for basal metabolism and for Ca²⁺ handling in E-C coupling, which are both components of the VO₂ intercept of VO₂ -PVA relation, were not significantly different from those of “Normal” and “Sham” groups.

Table 3.

Variables of LV energetics

		VO2-PVA relation		VO2 per minute (μl O2 · min-1 · g-1)
Group	n	Slope (X10-2μl O2 · mmHg-1 ml-1)	VO2-intercept (μl O2· beat-1 · g-1)	Basal Metabolism	E-C Coupling
Normal	6	1.13±0.29	0.303±0.043	29.0±3.2	60.3±13.4
Sham	6	1.26±0.24	0.348±0.026	29.6±3.4	71.8±5.3
Banding	6	2.61±0.82a.b	0.364±0.040	33.8±3.1	72.4±11.8
Banding + βgal	5	2.19±0.25a,b	0.310±0.044	35.1±3.4	56.7±13.5
Banding + saline	5	2.11±0.40a,b	0.304±0.036	28.6±3.3	62.7±11.8
Banding + SERCA	6	1.09±0.23c,d,e	0.377±0.064	33.4±2.7	78.6±17.9
Banding + Parv	5	1.37±0.34c,d,e	0.350±0.032	32.0±4.1	69.4±7.1

All data are shown as mean ± SD. VO₂, myocardial oxygen consumption per beat; PVA, systolic pressure-volume area;

E-C, excitation-contraction; n, number of hearts. VO₂ per minute for basal metabolism was measured in the hearts under KCl-induced arrest. VO₂ per minute for E-C coupling was obtained by subtracting VO₂ per minute for basal metabolism from VO2 per minute for mechanically unloaded (i.e., free of balloon water) contraction.

^aP < 0.05 compared to “Normal” group.

^bP < 0.05 compared to “Sham” group.

^cP < 0.05 compared to “Banding” group.

^dP < 0.05 compared to “Banding + βgal” group.

^eP < 0.05 compared to “Banding + saline” group.

3.4. ESP_mLVV in response to calcium

Table 4 shows changes in end-systolic pressure observed at a mLVV (ESP_mLVV) in response to Ca²⁺ infusion. ESP_mLVV prior to Ca²⁺ infusion in “Banding+SERCA” and “Banding+Parv” groups were significantly higher than those in “Banding”, “Banding+βgal” and “Banding+saline” groups. ESP_mLVV was gradually increased as the infusion rate of Ca²⁺ solution was increased step-wise from 2 to 6 ml/h. In “Banding+SERCA” and “Banding+Parv” groups, the maximal increase in ESP_mLVV in response to Ca²⁺ solution (6 ml/h) were similar to those in “Normal” and “Sham” groups, and were much higher than those in “Banding”, “Banding+βgal” and “Banding+saline” groups. In some experiments, we infused dobutamine solution into the coronary perfusion tubing at 2-4 ml/h of infusion rate after the Ca²⁺ infusion. The changes in ESP_mLVV in response to dobutamine were similar to those to Ca²⁺ in all groups (data not shown).

Table 4.

ESP at midrange left ventricular volume in response to Ca ²⁺ infusion

Group	n	ESPmLVV before Ca2+ infusion (mmHg)	ESPmLVV during Ca2+ infusion (mmHg)	Increase in ESPmLVV(mmHg)
Normal	6	114.2 ± 26.0	168.2 ± 24.8	54.0 ± 18.1
Sham	6	144.5 ± 22.7	199.8 ± 21.7	55.3 ± 12.2
Banding	6	86.1 ± 26.7	98.8 ± 28.9a,b	12.7 ± 4.6a,b
Banding + βgal	5	94.6 ± 26.8	115.0 ± 34.8a,b	20.3 ± 9.8a,b
Banding + saline	5	91.1 ± 21.5	114.7 ± 18.6b	23.6 ± 7.1a,b
Banding + SERCA	6	154.1 ± 58.2c,d,e	198.8 ± 62.6c,d,e	44.7 ± 14.7c,d,e
Banding + Parv	5	170.6 ± 26.1c,d,e	231.8 ± 34.1a,c,d,e	61.2 ± 11.2c,d,e

All data are shown as mean ± SD.

ESPmLVV, end-systolic pressure observed at midrange left ventricular volume (0.05 ml of balloon volume); n, number of hearts.

ESPmLVV during Ca²⁺ infusion was obtained in a steady state of hearts after intracoronary infusion of 1% CaCl2 at a 6 ml/h infusion rate for at least 4 minutes.

^aP < 0.05 compared to “Normal” group.

^bP < 0.05 compared to “Sham” group.

^cP < 0.05 compared to “Banding” group.

^dP < 0.05 compared to “Banding + βgal” group.

^eP < 0.05 compared to “Banding + saline” group.

3.5. LV contractility

The eEmax at mLVV, an index of LV contractility, in “Banding+SERCA” and “Banding+Parv” groups was significantly higher than that in “Normal”, “Sham” and 3 other control aortic banding groups (Fig. 2). In the preliminary experiments, 3 aortic banded rats underwent LV mechanical and energetic studies 45 days after recombinant AAV.SERCA transfer. As shown in Fig. 2, “Banding+AAV.SERCA” group showed the high LV contractility similar to that found in “Banding+SERCA” and “Banding+Parv” groups. On the other hand, “Banding”, “Banding+βgal” and “Banding+saline” groups showed the lower LV contractility.

Fig. 2.

Comparison of eEmax at midrange LV volume, an index of LV contractility, among all groups. *P<0.05 compared to “Normal”, “Sham”, “Banding”, “Banding+βgal” and “Banding+saline” groups. n=6 in “Normal”, n=6 in “Sham”, n=6 in “Banding”, n=5 in “Banding+βgal”, n=5 in “Banding+saline”, n=6 in “Banding+SERCA”, n=3 in “Banding+AAV.SERCA”, n=5 in “Banding+Parv”.

3.6. LV energetcs; oxygen cost of LV contractility

Figure 3A shows representative relations between VO₂ for Ca²⁺ handling in E-C coupling and eEmax at mLVV during Ca²⁺ inotropism run in “Sham”, “Banding” and “Banding+SERCA” hearts. These 3 distinct linear relations had the different slopes, which mean the different oxygen costs of LV contractility. In some experiments, the intracoronary dobutamine infusion was performed after the Ca²⁺ infusion in the same heart preparation. There was a fairly good correlation between the oxygen costs of LV contractility in response to Ca²⁺ and to dobutamine (Fig. 3B). The oxygen cost of LV contractility for Ca²⁺ in “Banding” group was about 3 times as high as that in “Normal” and “Sham” groups (Fig. 3C). In “Banding+SERCA”, “Banding+AAV.SERCA” and “Banding+Parv” groups, the oxygen costs of LV contractility were as low as those in “Normal” and “Sham” groups, but in “Banding+βgal and ”Banding+saline“ groups the oxygen costs remained as high as that of ”Banding“ group.

Fig. 3.

(A) Linear relations between Ca²⁺ handling VO2 and eEmax at midrange LV volume during Ca²⁺ inotropic run in “Sham” (■), “Banding” (▲) and “Banding+SERCA” (●) hearts. Slope of these linear relations means oxygen cost of LV contractility. (B) Correlation between O2 costs for Ca²⁺ and for dobutamine. In 17 heart preparations, dobutamine (78 μM) inotropic run was performed after Ca²⁺ inotropic run. An equation of regression line is f(x) = 0.93x + 1.8X10^-6, r = 0.96. (C) Comparison of oxygen costs of LV contractility among all groups. *P<0.05 compared to “Normal” and “Sham” groups. ^#P<0.05 compared to “Banding”, “Banding+βgal” and “Banding+saline” groups. n=6 in “Normal”, n=6 in “Sham”, n=6 in “Banding”, n=5 in “Banding+βgal”, n=5 in “Banding+saline”, n=6 in “Banding+SERCA”, n=3 in “Banding+AAV.SERCA”, n=5 in “Banding+Parv”.

3.7. SERCA2a & parvalbumin protein expression

We examined the protein expression of SERCA2a in the heart preparations used for the analysis of mechanical and energetic function. As shown in Figure 4, there is a decrease in SERCA2a expression in the failing hearts compared to sham. The overexpression of SERCA2a either by adenovirus or AAV1 restores SERCA2a protein levels in the failing hearts. Gene transfer of parvalbumin induces a robust expression in the hearts infected.

Fig. 4.

Immunoblots of SERCA2a and parvalbumin from left ventricular tissues of various groups.

4. Discussion

In this study, using a unique technique for measuring LV mechanoenergetics in the cross-circulated heart preparations, we found that cardiac gene transfer of Ca²⁺ handling proteins (SERCA2a and parvalbumin) can restore not only mechanical but also energetic function in pressure overload-induced failing hearts.

4.1. Mechanical function and gene transfer

In the failing rats, we found a downward shift of ESPVR, a decreased ESP_0.1 and deceased eEmax at mLVV compared with “Normal” and “Sham” rats. Furthermore, the logistic time constant of isovolumic relaxation and EDP_0.1 were found to be increased in these failing rats. Thus the aortic banded rats used in this study showed both LV systolic and diastolic dysfunction. The central mechanisms for systolic dysfunction in this rodent model of heart failure have been reported to be: decreased SERCA2a expression [17], increased sarcolemmal NaCaX expression [19], and the transition of cardiac myosin isoform components from V₁ with higher ATPase activity to V₃ with lower ATPase activity [20]. The decreased SERCA2a and/or the increased NaCaX can reduce the Ca²⁺ content in the SR and limit the Ca²⁺ release from the SR, leading to diastolic increases in calcium and general intracellular calcium overload [19]. V₁ is known to be associated with an increased shortening velocity of the cardiac fibers and V₃ with a slower shortening velocity [21], although in aortic banded rats, the improved contractile function by clenbuterol, a selective β₂-adrenergic agonist, was not related to changes in myosin isoform components [3]. On the other hand, the central mechanisms that cause diastolic dysfunction in this rodent model of heart failure are due to decreased SERCA2a expression and increase in cardiac stiffness due to enhanced collagen and fibrosis [3]. However, in this study the contractile dysfunction found in the “Banding” hearts (ESPVR, ESP_0.1, EDP_0.1, and time constant of isovolumic relaxation) could be completely corrected by SERCA2a gene transfer, indicating that the decreased SERCA2a expression plays a key role in both systolic and diastolic dysfunction. Furthermore, similar correction of contractile dysfunction could be accomplished by parvalbumin gene transfer as reported in normal and aged rats [11,22]. In senescent rat cardiomyocytes, introduction of parvalbumin, that functions as a Ca²⁺ sink due to its Ca²⁺ affinity and enhances relaxation in skeletal muscle, increased both the rate of Ca²⁺ transient decay and the rate of myocyte re-lengthening [23]. This in vitro study provides the cellular mechanism for improved diastolic function by parvalbumin gene transfer in the present in vivo study. On the other hand, the possible mechanism for improved systolic function by the parvalbumin gene transfer is as follows; the parvalbumin-induced decline in intracellular free Ca²⁺ during relaxation attenuates the intracellular Ca²⁺ overload and may improve the impaired Ca²⁺ handling in the present failing hearts. Finally, the prominent eEmax at mLVV could be induced by the SERCA2a or parvalbumin gene transfer in the aortic banded hearts. The high LV contractility, as indicated by high ESP_0.1 and high eEmax at mLVV, seems due mainly to both the hypertrophied LV free wall and the enhanced Ca²⁺ handling by overexpression of the SERCA2a or parvalbumin.

In the failing hearts, the increase in ESP_mLVV in response to Ca²⁺ infusion was much lower than that of “Normal” and “Sham” hearts. This blunted inotropic response to Ca²⁺ is most likely caused by multiple factors including abnormal Ca²⁺ handling, deficient production of cyclic AMP [24], along with a decrease in energy reserve via a deficient creatine kinase reaction [25]. Both SERCA2a and parvalbumin gene transfer was capable of completely reversing the inotropic response to Ca²⁺ (i.e., contractile reserve), indicating that in the aortic banded hearts the abnormal Ca²⁺ handling is a central cause of the lowered contractile reserve and regardless how calcium is decreased intracellularly, there is a beneficial response.

4.2. LV energetic function and gene transfer

In the failing hearts, the slope of the VO₂-PVA relation, which is an index of the O₂ cost of PVA, i.e., the O₂ cost of total mechanical energy of LV contraction [26], was about twice as high as that of “Normal” and “Sham” hearts. A similar steeper slope of the VO₂-PVA relation was reported in hyperthyroid rabbit hearts which actually have increased V₁/V₃ ratio [27]. In contrast, in hypothyroid [15] and diabetic [12] rat hearts with decreased V₁/V₃ ratio, the slope remained unchanged. Thus the inconsistent relationship between the slope and the V₁/V₃ ratio suggests that a change in myosin isoform components, i.e., a change in myosin ATPase activity, does not directly affect the contractile efficiency, which is the reciprocal of slope of the VO₂-PVA relation [26]. The contractile efficiency reflects the chemo-mechanical energy transduction efficiency of the contractile machinery. Therefore, in the aortic banded hearts, the high O₂ cost of total mechanical energy, shown by the steeper slope of the VO₂-PVA relation, indicates that energy wasting in the chemomechanical energy transduction is present. In the pressure overload-induced hypertrophied myocardium, the slope of relation between VO₂ and isometric developed tension was increased [28,29], and the increased VO₂ was due to the increased nonphosphorylating (state 4) mitochondrial respiration linked to Ca²⁺ transport [28], while in volume overload-induced hypertrophied myocardium, the slope was not different from that of normal myocardium [30]. Therefore, It seems likely that in the aortic banded hearts, the increased slope of the VO₂-PVA relation is attributable to the increased VO₂ induced by the increased nonphosphorylating mitochondrial respiration. This high slope could be completely improved by gene transfer of SERCA2a or parvalbumin. We have speculated on one possible mechanism for this; the overexpression of SERCA2a or parvalbumin restores the increased cytosolic free Ca²⁺ o normal levels and improves the enhanced Ca²⁺ transport to mitochondria, leading to the normal nonphosphorylating respiration of mitochondria. On the other hand, the VO₂-intercept (i.e., PVA-independent VO₂) of the VO₂-PVA relation, that reflects VO₂ for non-mechanical works consisting of Ca²⁺ handling during E-C coupling and basal metabolism [26], did not differ among all the groups studied. Furthermore, there was no significant difference in minute VO₂ for Ca²⁺ handling and basal metabolism among all the groups, showing that these minute VO₂ are affected neither by cardiac hypertrophy nor by adenoviral gene transfer. In addition, the unchanged VO₂ for Ca²⁺ handling found in the aortic banded hearts suggests that SERCA2a, albeit down-regulated, can exert its function normally because of a lower level of cytosolic free Ca²⁺ during diastole in steady state of mechanically unloaded and Ca²⁺ unloaded hearts.

The most important finding of this study is that the O₂ cost of LV contractility, defined as the slope of relation between Ca²⁺ handling VO₂ and eEmax, was increased in the failing hearts, and this increased O₂ cost could be restored to normal levels by short-term and long-term gene transfer of SERCA2a or parvalbumin. The O₂ cost of LV contractility is thought to indicate the energy cost of Ca²⁺ handling during E-C coupling in failing hearts where the Ca²⁺ responsiveness of myofilaments remains unchanged [31]. There seems three possible mechanisms for the increased O₂ cost of LV contractility, which means the energy wasting in Ca²⁺ handling, in the aortic banded hearts. First, the molar coupling ratio of calcium to ATP in SERCA2a was decreased due to increased Ca²⁺ permeability of the SR membrane [32]. Such SERCA2a dysfunction would need more ATP to lower cytosolic Ca²⁺ during relaxation. Second, abnormal Ca²⁺ leak through ryanodine receptor (RyR), caused by RyR conformational change due to a partial loss of RyR-bound FK506-binding protein, was found in a canine model of heart failure [33]. This Ca²⁺ leak via RyR will elevate basal cytosolic Ca²⁺ levels during diastole, leading to the futile Ca²⁺ handling. However, the above-mentioned mechanisms cannot fully explain the increased O₂ cost of LV contractility because of the down-regulated SERCA2a expression in our aortic banded hearts. Finally, cardiac NaCaX expression was reported to be up-regulated in heart failure [19,34,35]. Although NaCaX consumes no ATP to remove cytosolic Ca²⁺ in exchange with influx Na⁺ on the basis of a stoichiometry of 3Na⁺:1Ca²⁺, the influx Na⁺ will be pumped out by Na⁺-K⁺-ATPase with a stoichiometry of 3Na⁺:2K⁺:1ATP, resulting in the net stoichimetry of 1Ca²⁺:1ATP [36]. On the other hand, SERCA2a removes cytosolic Ca²⁺ on the basis of a stoichiometry of 2Ca²⁺:1ATP. Therefore, the Ca²⁺ extrusion via NaCaX leads to twice increase in energy expenditure compared with the Ca²⁺ uptake by SERCA2a. Thus it seems most likely that the energy wasting in Ca²⁺ handling is induced mainly by the enhanced NaCaX activity. Nevertheless, overexpression of SERCA2a or parvalbumin was capable of restoring the high O₂ cost of LV contractility to normal levels in the aortic banded hearts. The enhanced expression of SERCA2a or parvalbumin appears to induce the decrease in the cytosolic free Ca²⁺ level during diastole and thereby the decrease in Ca²⁺ extrusion via NaCaX, leading to the decrease in the energy cost of Ca²⁺ handling, namely, the decrease in the O₂ cost of LV contractility.

4.3. Therapeutic implications

In patients with congestive heart failure (CHF), inotropic agents improve contractility and hemodynamics in the short term. However, they increase VO₂, and subsequently induce an energetic imbalance, followed by arrhythmias and worsening survival. Therefore, long-term inotropic interventions have been shown to increase morbidity and mortality in patients with CHF [37]. In this study, gene transfer of SERCA2a or parvalbumin was found to improve the energy wasting both in chemomechanical energy transduction and in Ca²⁺ handling during E-C coupling in pressure-overload failing hearts. In a previous study [9], our group had already shown improvement in mortality and energy reserve (measured by NMR) in the same model of heart failure following short-term gene transfer of SERCA2a. Therefore, targeting Ca²⁺ handling in CHF may provide great advantage of efficient energy utilization over standard inotropic agents.

4.4. Limitations of the study and future directions

In the present failing hearts, correction of the energetic dysfunction by SERCA2a overexpression was accomplished, in a long-term transgene expression system of AAV1-based vectors as well as in a short-term transgene expression system of adenoviral vectors. To further confirm this finding, large animal studies are also required to verify these provocative results, because the contribution of SERCA2a to the lowering of cytosolic Ca²⁺ levels varies between rodents and large mammals. In humans, about 75% of the Ca²⁺ is removed by SERCA2a, whereas in rodents 92% is removed by SERCA2a [1]. However, overexpression of SERCA2a was capable of improving contractile function in failing human cardiomyocytes [38]. Therefore, SERCA2a overexpression is expected to be useful for improving LV energetic function in failing human hearts. Taken together, large animal studies using AAV vectors are indispensable for leading to clinical trials of gene therapy for CHF.

4.5. Conclusions

High O₂ costs of total mechanical energy and of LV contractility in the aortic banded hearts indicate the energy wasting both in chemomechanical energy transduction and in Ca²⁺ handling during E-C coupling. The gene transfer of Ca²⁺ handling proteins (SERCA2a and parvalbumin) was capable of correcting both the high O₂ costs. Thus the gene transfer of the Ca²⁺ handling proteins transforms the inefficient energy utilization into a more efficient state, and improves not only mechanical but also energetic function in pressure-overload failing hearts. Gene transfer of Ca²⁺ handling proteins may be a potential therapeutic strategy with the benefit of efficient energy utilization.