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Cardiac-specific overexpression of a high Ca²⁺ affinity mutant of SERCA2a attenuates in vivo pressure overload cardiac hypertrophy¹

HIROYUKI NAKAYAMA^*, KINYA OTSU^*2, OSAMU YAMAGUCHI^*, KAZUHIKO NISHIDA^*, MOTO-O DATE^*, KENICHI HONGO, YOICHIRO KUSAKARI, TOSHIHIKO TOYOFUKU^*, SHUNGO HIKOSO^*, KAZUNORI KASHIWASE^*, TOSHIHIRO TAKEDA^*, YASUSHI MATSUMURA^¶, SATOSHI KURIHARA, MASATSUGU HORI^* and MICHIHIKO TADA^*

^* Department of Internal Medicine and Therapeutics,
^¶ Department of Medical Information Science, Osaka University Graduate School of Medicine, Suita, Osaka; and
Department of Cardiology and
Department of Physiology, Jikei University School of Medicine, Tokyo, Japan

²Correspondence: Department of Internal Medicine and Therapeutics, Box A8, Osaka University Graduate School of Medicine, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: kotsu{at}medone.med.osaka-u.ac.jp

SPECIFIC AIMS

The cardiac Ca²⁺ ATPase (SERCA2a) of the sarcoplasmic reticulum (SR) plays a dominant role in lowering cytoplasmic calcium levels during relaxation. The aim was to examine whether modulation of SERCA2a activity results in the attenuation of cardiac hypertrophy and enhancement of contractility.

PRINCIPAL FINDINGS

1. Overexpression of the mutant SERCA2a in hearts leads to increased SERCA2a activity
We generated cardiac-restricted transgenic mice (TG) expressing the mutant SERCA2a (K397/400E), which disrupts the functional association with phospholamban (PLN). The maximum velocity of Ca²⁺ uptake (V_max) for TG hearts tended to be higher (35.91±1.61 nmol of Ca²⁺·mg^-1·min^-1 for nontransgenic littermate controls (NLC), 41.19 ± 2.86 nmol of Ca²⁺·mg^-1·min^-1 for TG), but the difference was not statistically significant (Fig. 1 A). However, the EC₅₀ value for Ca²⁺ decreased significantly compared with the NLC (0.39±0.02 µM for NLC, 0.24±0.02 µM for TG) (Fig. 1A, B ).

View larger version (16K): [in this window] [in a new window]   Figure 1. Initial rates of SR Ca2+ uptake. Cardiac homogenates prepared from mutant SERCA2a TG (n=4) and NLC (n=4) were assayed for Ca2+ dependence of Ca2+ transport (A). B) Percentage of the maximal Ca2+ uptake rates. Values are mean ± SE.

2. Increased contraction and relaxation with enhanced Ca²⁺ transient
Cardiomyocytes isolated from TG hearts exhibited a significant increase in Ca²⁺ transient amplitude relative to NLC hearts. Peak systolic [Ca²⁺]_i tended to increase and diastolic [Ca²⁺]_i at rest to decrease in TG hearts, but the changes were not significant. After reaching peak values, the Ca²⁺ transients declined more rapidly in the mutant SETCA2a TG than in their control cells. The time constant for Ca²⁺ decay significantly decreased by 35% in TG cardiomyocytes. Edge detection records from isolated myocytes showed an increase in the percentage of cell shortening in TG relative to their NLC. The time-to-peak shortening was identical for control and TG. The time needed to reach 75% of maximum relengthening for NLC and TG myocytes averaged 238 and 147 ms, respectively.

There were no significant differences in peak systolic or end diastolic left ventricular pressure or heart rate for TG vs. NLC. Both the maximum and minimum first derivatives of the left ventricle (LV) pressure significantly increased in TG hearts compared with their NLC hearts.

4. Attenuation of left ventricular hypertrophy induced by pressure overload
TG were subjected to pressure overload by transverse thoracic aortic constriction (TAC) operation. Cardiac hypertrophy in TAC-operated TG hearts was apparently less than their TAC-operated NLC (Fig. 2 A). Across a wide range of systolic pressure gradients, the LV weight-to-tibia length ratio (LVW/TL) was lower for TG than for their NLC (Fig. 2B ). The increase in LVW/TL by TAC was 33% in TG and 62% in NLC. The increase in ANF expression induced by TAC was significantly attenuated in the mutant SERCA2a TG compared with their NLC (Fig. 2C ). The skeletal -actin mRNA level was also attenuated, but not significantly, in the mutant SERCA2a TG hearts. There were no significant differences in TAC-induced decreases in SERCA2a and PLN mRNA levels for the mutant SERCA2a TG compared with their NLC.

View larger version (17K): [in this window] [in a new window]   Figure 2. Characteristics of mutant SERCA2a TG after induction of pressure overload. Mice were killed 7 days after TAC and their hearts were removed. A) Hearts were examined by hematoxylin-eosin stain. B) Index of the left ventricular mass (LVW/TL) is plotted against the systolic pressure gradient produced by TAC for TG (n=8) and their NLC (n=7). White and black circles represent TG and NLC. C) Left ventricle ANF, skeletal -actin, SERCA2a, and PLN mRNA quantitation by dot blot analysis. Data represent fold induction relative to sham-operated mice and are shown as the mean ± SE (n=3). *P < 0.05 mutant SERCA2a TG vs. their NLC (unpaired t test).

5. Selective inhibition of Ca²⁺-dependent PKC isozymes
Pressure overload also increased particulate levels of PKC, ß1 (Ca²⁺-dependent PKC) and (Ca²⁺-independent PKC) in NLC compared with sham-operated controls (2.31-, 2.32-, and 2.15-fold increases, respectively). Increases in PKC and ß1 in the particulate fraction were significantly attenuated in TG hearts (1.67- and 1.47-fold increase vs. sham-operated TG, respectively). However, there were no significant differences in particulate levels of PKC between TAC-operated TG and NLC.

CONCLUSION AND SIGNIFICANCE

In this study, overexpression of the mutant SERCA2a was associated with a significant increase in Ca²⁺ affinity of SERCA2a in microsomes isolated from hearts (Fig. 2) , suggesting that overexpressed SERCA2a integrated into SR membranes in the TG hearts and seems to work as a high-affinity mutant in the TG hearts. The enhanced SR function resulted in increased contraction and relaxation in isolated cardiomyocytes and intact mice (Fig. 3 ).

View larger version (24K): [in this window] [in a new window]   Figure 3. Characterization of the mutant SERCA2a transgenic mice. Overexpression of the high Ca2+ affinity mutant SERCA2a resulted in attenuation of pressure overload-induced cardiac hypertrophy and enhancement of cardiac contractility. Thus, modulating SERCA2a-PLN interaction may lead to the development of novel therapeutic strategies against cardiac hypertrophy that have both anti-hypertrophic and positive inotropic effects.

When wild-type SERCA2a TG were subjected to pressure overload, the magnitude of LV hypertrophy was similar in wild-type SERCA2a TG and their NLC. This suggests that the attenuation of cardiac hypertrophy observed in the mutant SERCA2a TG might be due to an increase in the affinity of SERCA2a for Ca²⁺.

Overexpression of the mutant SERCA2a resulted in attenuation of the increase in Ca²⁺-dependent PKC and ß1 but not Ca²⁺-independent PKC in the particulate fractions. Thus, our findings with previous studies of wild-type TG suggest that the peak systolic [Ca²⁺]_i and overall kinetics of Ca²⁺ transient are not related to cardiac hypertrophy and activation of Ca²⁺-dependent cardiotrophic signals such as PKC and PKCß1, but that the kinetics of Ca²⁺ cycling at a low Ca²⁺ concentration is critical for activation of the Ca²⁺-dependent signals leading to cardiac hypertrophy. Attenuation of TAC-induced cardiac hypertrophy in the mutant SERCA2a TG might be due at least in part to the inhibition of Ca²⁺-dependent PKC activation by the enhanced Ca²⁺ lowering capacity.

Previous studies have demonstrated important roles for Ca²⁺-dependent signaling pathways in hypertrophic responses. On the other hand, muscle contractility is regulated by [Ca²⁺]_i. Thus, the attenuation of cardiac hypertrophy and enhancement of cardiac function appear to be incompatible phenomena. In this study, however, cardiac-specific overexpression of the high Ca²⁺ affinity mutant SERCA2a resulted in both attenuation of pressure overload-induced cardiac hypertrophy and enhancement of cardiac contractility.

Our results indicate that modulating SERCA2a-PLN interaction may lead to the development of novel therapeutic strategies against cardiac hypertrophy that have both anti-hypertrophic and positive inotropic effects. However, long-term effect of overexpression of the high Ca²⁺ affinity SERCA2a mutant on the onset and progression of heart failure after cardiac hypertrophy has to be examined in the future.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0474fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.02-0474fje

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