INTRODUCTION

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TYPE II MYOSIN, a hexamer composed of two myosin heavy chains (MyHCs), each associated with two types of light chains (MyLCs), is the molecular motor of all muscle cells. (17). At least seven different genes coding for MyHCs and seven genes coding for MyLCs exist (15, 20, 24). Splicing of exon 39 generates SM1 (204 kDa), and inclusion of exon 39 yields SM2 (200 kDa) (1, 10, 19, 21, 23, 27). Splicing of exon 5b generates the A forms; its inclusion generates the B forms (10, 19, 21, 23).

Smooth muscle MyHC B (SMB) revealed an approximately twofold higher actin-activated Mg²⁺-ATPase activity, attachment time (14), and actin filament movement velocity in the in vitro motility assay than smooth muscle MyHC A (SMA; Refs. 13, 22). Recently, an SMB knockout mouse model confirmed higher force generation and cross-bridge cycling kinetics of smooth muscle preparations in the presence of SMB (2).

The phasic smooth muscle tissue from the antrum region of the stomach (8), urinary bladder (11, 27, 28), esophageal body (25), intestine (27), trachea, and airway (16) contains predominantly the SMB isoform, whereas smooth muscle cells from myometrium (27), urethra (11), lower esophageal sphincter (25), fundus region of the stomach (8), the corpus cavernosum (7), aorta (6, 27), and vena cava (27) contain predominantly the SMA isoform. In contrast, precapillary arterioles expressed considerable amounts of the B form (6, 12, 16, 26).

Normotensive rats revealed a higher fraction of precapillary arterioles with detectable SMB expression than spontaneously hypertensive rats (SHRs) of the stroke-prone strain (26). To investigate whether this difference is of genetic origin or represents an adaptive response to chronically elevated blood pressure and cardiac hypertrophy, we determined the expression of SMB in cardiac vessels in two experimental models of hypertension-induced cardiac hypertrophy [1 clip, 1 kidney (1C1K); ANG II minipump].

	MATERIALS AND METHODS

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Surgical procedures and tissue preparations. Renovascular hypertension was induced by unilateral stenosis of the left renal artery (0.22-mm silver clip) in 6-wk-old male Sprague-Dawley (SD) rats (1C1K). In matched sham-operated control animals, the renal artery was partially exposed by removal of rounding tissue. After 3 days, blood pressure was measured in the femoral artery between 9 AM and 11 AM in the freely moving animal (P23Db Statham pressure transducer, 4600 Gould pressure processor). Animals were killed 6 wk later. For continuous subcutaneous ANG II infusion, 3-mo-old male SD rats received ANG II by osmotic pump for 7 days (Alzet, model 2001, Alza) with an infusion rate of 250 ng ANG II · kg¹ · min¹. Blood pressure was measured by the tail-cuff method under light and short (2 min) ether anesthesia.

Immunofluorescence microscopy. Preparation of cryostat sections (5 µm thickness), fixation, and immunolabeling were performed as recently described (26) using a rabbit anti-SMB antibody (a25K/50K; cf. Ref. 22) and mouse anti-smooth muscle-actin antibody (aSM-actin, Boehringer, Germany). Primary antibodies were visualized as described previously (26). Nuclei were stained simultaneously by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma). Immunolabeling was evaluated with an Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany) using appropriate filter systems. Micrographs were taken with an MC 100 automatic camera (Zeiss). All aSM-actin-positive and a25K/50K-positive vessels in a section were counted (2 sections/ventricle; 6 animals/group). The fraction of a25K/50K-positive vessels was expressed in percentage of vessels detected by aSM-actin staining.

Statistical evaluation. Results are expressed as means ± SE. Significance analysis was performed with the Student's t-test.

	RESULTS

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Expression of SMB was analyzed by immunofluorescence microscopy of cryostat sections of rat left ventricle using a double-labeling method (cf. Ref. 26). The specific aSM-actin antibody was used to identify cardiac vessels, and the a25K/50K antibody was used for simultaneous detection of the SMB isoform. Cardiomyocytes did not react with these antibodies (Fig. 1).

View larger version (68K): [in this window] [in a new window]   Fig. 1.   Immunofluorescence micrograph of a left rat ventricle double-labeled with a25K/50K (A) and anti-smooth muscle-actin (aSM-actin) antibodies (B). Primary antibodies were visualized with species-specific secondary antibodies conjugated with Cy3 (A) and DTAF (B). Most small vessels of ventricular tissue were labeled with both antibodies, whereas all large vessels were only labeled with the aSM-actin antibody. Arrows mark a small vessel without smooth muscle myosin heavy chain B (SMB).

In normal adult rats, the labeling pattern differs in blood vessels of different size. Most small vessels with wall thickness <13 µm and lumen diameter <18 µm of normal left ventricles were labeled with a25K/50K (Fig. 1A) and aSM-actin antibodies (Fig. 1B). Staining of nuclei with DAPI showed that the SMB-positive vessels were surrounded by a single layer of smooth muscle cells (precapillary arterioles) (not shown). Large vessels were characterized by a wall thickness of 13 µm and a lumen diameter of 18 µm. In all sections the percentage of large vessels represented barely 2% of all blood vessels. All large vessels reacted only with aSM-actin but not with a25K/50K antibody (Fig. 1B).

In another set of experiments, we compared the fraction of SMB-positive precapillary arterioles, i.e., blood vessels with wall thickness <13 µm and lumen diameter <18 µm and single smooth muscle cell layer, of the left ventricle of normotensive with experimentally hypertensive rats (1C1K and ANG II treated). Similar to control rats, only precapillary arterioles of hypertensive rats reacted with a25K/50K. The fractions of SMB-positive vessels ranged between 66.9 and 71.5% of all precapillary arterioles investigated (Table 1). This was not significantly different compared with the corresponding control animals (range 62.4-67.8%; Table 1).

Blood pressure of 1C1K and ANG II animals was significantly higher (P < 0.01) compared with the corresponding controls (Tables 2 and 3). In both experimental hypertension models, heart weight and the heart-to-body weight ratio were significantly (P < 0.05) higher than the respective control rats (Tables 2 and 3).

	DISCUSSION

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In a previous study we reported a significantly lower fraction of SMB-expressing ventricular precapillary arterioles in SHRs (26). To determine whether this difference is genetically determined or represents an adaptive response to hypertension and hypertrophy, we investigated the expression of SMB in cardiac vessels in two experimental models of chronic hypertension, namely in a 1C1K model of renal artery stenosis and in an ANG II minipump model. Between 62.4 and 67.8% of cardiac precapillary arterioles expressed SMB in control SD rats. This is in accordance with recent observations in Wistar-Kyoto rats (26). Both occlusion of renal artery, which is known to activate the renin-angiotensin system, as well as chronic infusion of the vasoconstrictor ANG II, induced hypertension and cardiac hypertrophy (cf. also Refs. 4 and 9). We found no different pattern of SMB expression in precapillary arterioles in experimental hypertension (range between 66.9 and 71.5% SMB-expressing precapillary arterioles) compared with normotensive controls. Therefore, we suggest that the abnormal SMB expression pattern in ventricular precapillary arterioles of SHRs is of genetic origin rather than the result of an adaptive response to chronically enhanced blood pressure and cardiac hypertrophy.

The physiological importance of SMB expression in most of the precapillary arterioles still has to be elucidated. Interestingly, precapillary arterioles are main targets of sympathetic regulation (3, 18). In addition, smooth muscle cells of the corpus cavernosum, which are tightly innervated with sympathetic nerves, expressed high amounts of SMB (7). It seems interesting to demonstrate whether cardiac blood vessels regulate their responsiveness to sympathetic innervation by differential expression of 5'-spliced smooth muscle MyHC isoenzymes. It could be demonstrated that actin-activated ATPase activity, velocity of actin filament movements in the in vitro motility assay, attachment time in the laser trap study, as well as isometric force development and shortening velocity of SMB isoforms are higher than SMA isoforms (2, 13, 14, 22). These functional features, therefore, could confer an accelerated contractile response of SMB-expressing precapillary arterioles to sympathetic stimulation.