Caveolin is a 21- to 24-kDa principal structural protein of caveolae, vesicular invagination of the plasma membrane (Lisanti et al., 1994). The mammalian caveolin family consists of caveolin-1, -2 and -3. Among them, caveolin-3 is muscle specific and found in skeletal, cardiac and smooth muscle (Song et al., 1996). Caveolin is known to be involved in cellular signaling cascades including G-proteins, sarcoplasmic reticulum (SR) kinase, nitric oxide (NO) synthase, epidermal growth factor, platelet-derived growth factor receptors and protein kinase C (Okamoto et al., 1998). Caveolin is also a substrate of the low M(r) protein-tyrosine phosphatase (Caselli et al., 2001). It is also shown that caveolin was down-regulated by chronic beta-adrenergic stimulation (Oka et al., 1997).

Caveolin is related with hypertrophy of skeletal and cardiac muscles. Myocardial caveolin-3 expression was decreased in 24-week-old SHR compared with age-matched Wistar-Kyoto (WKY) rats and 4-week-old Spontaneously Hypertensive Rat (SHR) (Toya et al., 2001). Rat neonatal cardiomyocytes had diminished hypertrophic response to overexpressing caveolin phenylephrine (Oka et al., 2001). Transgenic overexpression of caveolin-3 in skeletal muscle induced a Duchenne-like muscular dystrophy in mice (Galbiati et al., 2000). On the contrary, loss of caveolin-3 expression resulted in another form of muscular abnormality, limb-girdle type muscular dystrophy (Minetti et al., 1998).

These findings may indicate that adequate regulation of caveolin level may be necessary for the maintenance of normal muscle physiology. Caveolin may be an inhibitor of some types of cardiac hypertrophy, although the specific functions of caveolin is unknown. The role of caveolin in adaptive physiological hypertrophy is unknown. Caveolin may possibly be involved in the physiological hypertrophy signaling and may decrease with transition into decompensated hypertrophy or cardiac failure.

The specific purpose of this study was to compare the signal transduction, including caveolin, of cardiac hypertrophy between the hearts from rats with voluntary exercise-induced physiological left ventricular hypertrophy (LVH) and those from rats with aortic constriction-induced pathological LVH.

Group Assignments
All the animal experiments were approved by the Animal Experimentation Review Board of the University of Tokyo. Male Wistar rats were assigned to 1 of the following 3 groups: (1) 10-week voluntary exercise training (EX, n=9), (2) sedentary condition (SED, n=9), and (3) 4-week ascending aortic constriction (AC, n=9).

Voluntary Exercise-Induced Physiological LVH (Eto et al., 2000)
Four-week-old rats transferred to our laboratory was randomly assigned into the voluntary exercise group and sedentary group. Ones in the voluntary exercise group were individually housed in the specially manufactured cage equipped with a controlled running wheel and a distance counter. We controlled the torque of the running wheel, not the rotation speed. Therefore, what we measured as the distance was a daily work rather than the exercise intensity. Rats exercised at their favourite time, at a speed and duration of their choice (EX). Age-matched sedentary control rats were also individually housed in ordinary cages (SED). Ten weeks later, the hearts were excised and the following biochemical analyses were performed.

Pressure-Overload Induced Pathological LVH
Ten-week-old rats were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg·kg^-1). A ligature was placed around the ascending aorta, and an 18-gauge needle was set parallel to it. Then, the needle was quickly removed, leaving the ascending aorta constricted to the diameter of the needle (Aoyagi et al., 1999). Four weeks later, the biochemical assessments were performed.

RNA Protection Activity
Total RNA was extracted from the left ventricular tissue with TRIZOL (GIBCO, Carlsbad, CA), and used in the following RNase protection assays with probes for alpha- and beta-myosin heavy chain (MyHC), sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a), atrial-type natriuretic peptide (ANP), brain-type natriuretic peptide (BNP), alpha-skeletal and alpha-cardiac actins, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. Pathological examination in our previous study (Eto et al., 2000) revealed no significant fibrosis in either of the LVH models. Therefore, the left ventricular crude homogenates for the RNase protection assay and the Western blot were considered to consist of mainly cardiac myocytes.

All plasmids for riboprobes were sequenced, and linearized at the 3'-end of the antisense strand with appropriate restriction enzymes. The StyI-digested rat GAPDH probe (pTRI-GAPDH-Rat, Ambion) protected a 134-bp fragment (complimentary to nucleotides 369-503 of Accession number X002310). The probe specific for the rat SERCA2a gene protected a 333-bp fragment of SERCA2a (3014-3347 nucleotides of X15635). The probe for rat MyHC was complimentary to the 3'-end of beta-MyHC mRNA; it protected a 300-bp fragment (5626-5925 nucleotides of X15939), but also hybridized to alpha-MyHC, and protected a 175-bp fragment (5656-5830 nucleotides of X15938). The probes specific for the rat ANP and BNP genes protected a 212-bp fragment and a 183-bp fragment of ANP (96-307 nucleotides of E00443) and BNP (246-428 nucleotides of M25297), respectively. The probes specific for the rat alpha-skeletal actin and alpha-cardiac actin genes protected a 195-bp fragment and a 100-bp fragment of alpha skeletal actin (2841-3037 nucleotides of V01218) and alpha cardiac actin (1111-1210 nucleotides of X80130), respectively. Riboprobes were labeled with [alpha-³²P] UTP using Maxiscript kit (Ambion, Austin, TX). The MyHC and the SERCA2a probes had a specific activity of ~0.5x10⁸ and ~1.0x10⁸ cpm/µg, respectively, and were used with 3 µg total RNA. The GAPDH probe (specific activity; ~2x10⁸ cpm/µg) was mixed in each sample as an internal control. After incubation at 42°C overnight, the RNA-probe mix was digested with RNase A/T1 (1:100, RPAII, Ambion, Austin, TX) at 37°C for 30 min, and protected fragments were separated on a 6% denaturing polyacrylamide gel. Radioactivity of each band was quantified by densitometry. Signals were corrected for background and were normalized to GAPDH levels.

Western Blot Analysis for Myocardial Caveolin Content
Preparation of tissue extracts was performed as previously described (Yonekura et al., 2000). Briefly, hearts were homogenized in a buffer containing 50mM Tris-HCl (pH6.8), 2% sodium dodecyl sulfate (SDS), 6% 2-Mercaptoethanol, 10% glycerol, and protease inhibitors, and separated by 15% SDS-polyacrylamide gel. The proteins were then transferred onto poly vinylidene difluoride (PVDF) membranes. The membranes were incubated in tris-saline buffer (TBS) containing 0.1% Tween-20, 5% non-fat dry milk, 5% bovine serum albumin for 1 hour at room temperature. The membranes were then treated with each primary antibody (anti-caveolin-1, anti-caveolin-2, and anti-caveolin-3 monoclonal antibodies (Transduction Laboratories, Lexington, KY)) for two hours followed by washing and incubation with a secondary antibody. The signals were detected by SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL), exposed to Hyperfilm electrochemiluminescence (ECL) (Amersham Pharmacia Biotech, Buckinghamshire, UK), and quantitated by a densitometer.

Statistical Analyses
Differences among the groups were compared by non-repeated measures ANOVA and Student-Newman-Keuls test. Data were expressed as the mean (SD). P < 0.05 was considered significant.

The EX rats ran 1.6 (1.1) km·day^-1, and developed LVH. LV weight (LVW) and the LVW to body weight ratio (LVW·BW-1) increased in The EX rats by 15% (p < 0.05) and 24 % (p < 0.05), respectively, compared with the SED rats. The LVW and LVW·BW-1 increased in the AC rats by 30 % (p <0.01) and 23 % (p < 0.01), respectively (Table 1).

Myocardial Gene Expression Regarding Hypertrophy Signaling (Figure 1)
The ascending aortic constriction surgery induced the myocardial expressions of alpha skeletal actin and natriuretic peptides, while SERCA2a mRNA level was decreased by 29% (p < 0.05). In contrast, the voluntary exercise training induced isozyme shift of MyHC from beta subtype to alpha subtype. SERCA2a mRNA level was increased by 50% by the exercise training. Neither ANP nor BNP was elevated in the EX group.

Myocardial Caveolin Content (Figure 2)
The amount of caveolin-3 expression in the EX group was approximately 11% higher than that in the control group (p < 0.05). In contrast, the amount of caveolin-3 expression of the AC group was approximately 13% lower than that in the SED group (p < 0.05). That is, caveolin-3 is up-regulated in physiological cardiac hypertrophy, but down-regulated in pathological cardiac hypertrophy. Caveolin-1 and caveolin-2 protein levels did not differ among the groups.

Left ventricular hypertrophy (LVH) induced by pathological hemodynamic overload can eventually result in maladaptive congestive heart failure (CHF). LVH is a strong predictor of subsequent cardiovascular events (Devereux et al., 1995). In contrast, the athletes' heart, or LVH without pathological change, results from properly designed endurance exercise training (Barry, 1986). Elucidating the underlying molecular mechanisms that distinguish adaptive hypertrophy from pathological hypertrophy is important for the development of therapies to prevent CHF. We previously showed that calcineurin was activated in the voluntary exercise-induced physiological LVH but not in the decompensated pressure-overload hypertrophy (Eto et al., 2000). The LVW/BW, LV end-diastolic dimension, and velocity-time integral of aortic ejection flow increased, without an increase in cardiomyocytes width as reported in literature (Schaible et al., 1987, Moore et al., 1993). Other than calcineurin, many molecules participating in signal transduction of cardiac hypertrophy have been reported with controversies in their roles. Those controversies may come from the diversity of cardiac hypertrophy itself. We believe that the presence of adaptive physiological hypertrophy has different signal transduction from the pathological hypertrophy. In order to further clarify the characteristics of the voluntary exercise-induced physiological LVH, we advanced our investigation into several other molecules participating in signal transduction of cardiac hypertrophy including caveolin. The roles of caveolin in cardiac hypertrophy process are also controversial (Woodman et al., 2002, Galbiati et al., 2000, Minetti et al., 1998). The current study is the first report that shows the involvement of caveolin in physiological adaptive hypertrophy.

Although roles of caveolin-3 in physiological cardiac hypertrophy are unknown, association of caveolin-3 with Na⁺/Ca²⁺ exchanger (Bossoyt et al., 2002) may somehow explain it.

Since we classified the voluntary exercise as physiological response, the forced exercise would be a more pure counterpart. However, the forced running in rats have not successfully attained enough degrees of running distance on resultant hypertrophy corresponding to those of the current voluntary running model. Improvement in experimental design of forced running is in our future plan.

The exercise rats voluntarily ran 1.6±1.1km/day. The variation of the exercise amount among the animals was quite large. Some rats seemed to like physical exercise and our running wheel, while other did not. Despite this wide variation in the exercise amount, ventricular hypertrophy response was relatively steady. Effects of physical exercise may have on upper threshold, above which no more hypertrophic stimulus occurres. If this is the care, the dose-response relationship may not be linear. Other possible explanations in dude negative effects of sedentary circumstance on cardiac growth, which may be released in the voluntary exercise group.