Myocardial connexin43 expression in left ventricular hypertrophy resulting from aortic regurgitation.

Intercellular conduction in the working myocardium of the mammalian heart is mediated by gap junctions composed of connexin43 or 45. Recently, it has been shown that myocardial connexin expression is malleable and may be altered with disease. To better understand myocardial conduction in left ventricular hypertrophy resulting from volume overload, we used indirect immunofluorescence microscopy to examine cardiac connexin43 expression in 10 New Zealand white rabbits with surgically induced aortic regurgitation (AR) and in 10 age-matched sham-operated controls. Animals were sacrificed at approximately 1 month or > or =2.5 years after operation. All AR animals developed eccentric hypertrophy; none evidenced heart failure. The heart-to-body weight ratios for the 1 month AR and control groups were 2.9+/-0.8 vs 1.8+/-0.2 g/kg (p < or = 0.01) while ratios for the > or =2.5 year AR and control groups were 2.4+/-0.3 vs 1.9+/-0.3 (p < or = 0.05). No significant differences in posterior wall thickness were found among any of the groups. Although the overall pattern of connexin43-like immunoreactivity was similar for all four groups, staining in the I month AR animals tended to be less than that of age-matched controls; staining was increased in the > or =2.5 year AR animals and was greater than control (p < 0.05), in which staining did not change with animal age. This disease duration-related increase differs from the long-term decrease in connexin43 expression associated with other forms of heart disease and suggests that alterations in connexin expression may play a role in the rhythm abnormalities commonly seen in AR.

Fibrosis, myocyte degeneration and heart failure in chronic experimental aortic regurgitation.

Myocardial fibrosis and myocyte degeneration have been reported in patients with chronic aortic regurgitation (AR), and may be related to the pathophysiology of congestive heart failure (CHF) in this disease. To define the relationship between myocardial histopathologic variations and CHF in chronic AR, we performed gross and microscopic evaluations of postmortem tissue from a rabbit model of chronic AR manifesting left ventricular (LV) responses to AR similar to those in humans. Moderate-to-severe chronic AR (echocardiographic regurgitant fraction = 52 +/- 13%) was induced by closed-chest aortic valve perforation in 11 New Zealand White rabbits; 5 control rabbits were sham operated. Six of the 11 AR rabbits died 1.5 +/- 0.8 years (range 0.6-2.8 years) after AR induction; all 6 had gross and histologic anatomic evidence of CHF at necropsy. The remaining 5 AR rabbits survived until sacrifice at 2.9 +/- 0.1 years of AR; none had pathologic evidence of CHF. Cardiac hypertrophy and the extent of LV fibrosis and myocyte necrosis all were greatest among the 6 AR CHF rabbits. No inflammatory response was apparent in any animal. Moderate-to-severe chronic experimental AR frequently results in CHF which is strongly associated with myocardial fibrosis and necrosis, without evidence of inflammation. These histopathologic variations may be pathophysiologically related to CHF development.

Myocardial collagen in cardiac hypertrophy resulting from chronic aortic regurgitation.

Myocardial fibrosis and abnormal myocardial collagen content are common in many forms of pathological cardiac hypertrophy, including that mediated by pressure overload. Recently, in an experimental animal model of chronic aortic regurgitation (AR), we found a strong relation between myocardial fibrosis and congestive heart failure development. To determine if these fibrotic lesions are composed of collagen, as they are in pressure overload, and to determine if potential preventive therapies should be developed similarly in both diseases, we assessed left ventricular collagen content at three time points after AR induction. Moderate to severe AR was induced in 19 New Zealand White rabbits by inserting a catheter through the carotid artery to perforate the aortic valve leaflets. Animals were killed (1) when they showed echocardiographically discernible systolic dysfunction or (2) if normal cardiac function continued, either early (1 month) or late (>3 years) after operation. Fourteen age-matched, sham-operated controls and seven normal unoperated rabbits also were studied. Collagen concentrations were determined biochemically by hydroxyproline measurement. Fibrosis was measured histologically using Mason's trichrome stain and the fibrous collagen-specific stain, Picro-Sirius Red. Our results show an age-related increase in left ventricular collagen concentration with no specific increase among animals with evidence of fibrosis. We conclude that, unlike pressure overload, volume overload produces fibrotic lesions not composed predominantly of excess collagen and that the therapy needed to prevent fibrosis may be different in these conditions. Further study is needed to define the chemical characteristics of the fibrous lesions and the pathophysiological importance of this finding.

Differential response to vesnarinone by cardiac fibroblasts isolated from normal and aortic regurgitant hearts.

Vesnarinone, a quinoline derivative with modest positive inotropic action, has been shown in several studies to benefit patients with clinical congestive heart failure. The cellular basis of its clinical benefit is not known, although the drug has several pharmacologic effects demonstrated both in isolated cardiac myocytes and in other noncardiac cell types. To investigate the possibility that the clinical benefit of vesnarinone is based, at least in part, on the inhibition of pathologic myocardial fibrosis, we examined its effects on cultured cardiac fibroblasts isolated from both normal and aortic regurgitant New Zealand White rabbit hearts. As in people, rabbits with moderate-to-severe aortic regurgitation often develop congestive heart failure that, at necropsy, is characterized by exuberant myocardial fibrosis. A dose-response curve was constructed with vesnarinone concentrations ranging from 10(-4 ) to 10(-9 ) mol/L. Cellular survival was decreased by exposure to nanomolar concentrations of drug but not at the higher doses tested. Fibroblasts isolated from normal hearts responded maximally at 10(-7 ) mol/L vesnarinone, whereas fibroblasts from aortic regurgitant hearts responded maximally at 10(-8 ) mol/L. These concentrations of drug are more than an order of magnitude lower than those believed to be associated with clinical benefit from earlier studies. Our results indicate that vesnarinone can suppress cardiac fibroblast proliferation and suggest that this action may be useful in therapies designed to prevent congestive heart failure in aortic regurgitation.

Antimyosin antibody imaging in experimental aortic regurgitation.

BACKGROUND: Fiber dropout and myocyte necrosis precede heart failure in experimental aortic regurgitation (AR). The current study aimed to determine whether this process can be detected by noninvasive scintigraphic imaging. METHODS AND RESULTS: 111In-labeled antimyosin antibody Fab fragment (1 to 1.5 mCi) (Myoscint) was administered to each of 34 New Zealand White rabbits: 11 early (3 to 5 weeks) after surgical AR induction; 9 late (98 to 128 weeks) after AR induction; 5 normal and 3 sham-operated age-matched with early AR; and 3 normal and 3 sham-operated age-matched with late AR. Echocardiographic fractional shortening was indistinguishable among control, early AR, and late AR groups. In vivo gamma camera imaging 24 and 48 hours after isotope administration, post-mortem heart activity determination (percentage injected dose per gram), and autoradiography were performed. At 24 and 48 hours, heart-to-lung counts-per-pixel ratios from in vivo images were greater (p < 0.05) in the late AR rabbits than in each of the three other groups. No significant differences were found when early AR and older or younger control rabbits were compared. Heart activity (percentage injected dose per gram) in late AR rabbits trended toward higher values than in age-matched control rabbits (p = 0.057), but in early AR it was indistinguishable from that in the corresponding control (p = 0.413, difference not significant). The autoradiographic endocardial/epicardial activity ratio in late AR rabbits was greater than in control and early AR rabbits (1.27 +/- 0.13 vs 1.06 +/- 0.09 and vs 1.13 +/- 0.10, respectively, p < 0.02). CONCLUSIONS: Whereas isotope uptake in late AR rabbits differed from that in control and early AR rabbits, systolic function was indistinguishable. Thus 111In-labeled antimyosin antibody imaging may permit noninvasive detection of AR-induced myocardial damage before functional deterioration.

Biodistribution and autoradiographic localization of I-125--labeled synthetic Peptide in aortic atherosclerosis in cholesterol-fed rabbits.

I-125 labeled SP4 is a synthetic oligopeptide derived from apolipoprotein B of low-density lipoprotein that has been shown to localized in atherosclerotic plaques in experimental animals. However, its biodistribution and mechanism of localization need to be further elucidated. Twenty-four cholesterol-fed (CF) and 20 normal (NL) New Zealand White rabbits were injected with I-125-SP4 and killed 15 to 30 min (6 NL; 6 CF) or 2 h (14 NL; 18 CF) later. We obtained aortic autoradiograms and activity concentrations (% injected dose/gm) in aortic segments and other tissues. The uptake of I-125-SP4 was higher in CF than in NL rabbits in all aortic segments (p < 0.05). I-125-SP4 was cleared rapidly in both CF and NL rabbits with 60 to 70% of the injected dose cleared from the blood by 1 h. No statistically significant differences in radiotracer biodistribution were observed between NL and CF rabbits although activity tended to be higher in the liver, gallbladder, and intestine in NL rabbits and in the kidney and spleen in CF rabbits. Silver grains were distributed mainly on foam cells of the fatty streaks in aortic microautoradiograms from two additional rabbits that had been injected with I-125-SP4. There were 23,518 plus minus 15,878 (SD) grains/mm(2) in fatty plaques but only 14,669 plus minus 11,035 grains/mm(2) in media muscle (p < 0.0001 [9 sections, 17 areas evaluated] in an atherosclerotic animal) in injected animals and 13,439 plus minus 5,565 grains/mm(2) in media muscle (two sections, four areas) in the normal control animals (NS versus media of atherosclerotic animal). I-125-SP4 specifically localizes in aortic atherosclerotic plaques in CF rabbits. There is no significant difference in tissue distribution between normal and CF rabbits except in the aorta. Preliminarily, it appears that the site of tracer uptake is on foam cells and this suggests the possibility of relative specificity for fatty plaque.

Cell-free incorporation of newly synthesized myosin subunits into thick myofilaments.

Although a substantial literature exists on the in vitro polymerization of purified myosin, little is known about native thick filament assembly, remodeling or turnover. We have recently described a cell-free system (Bouche et al., 1988) to examine the interactions between thick filaments and soluble, newly synthesized myofibrillar proteins. In the present manuscript we describe our studies on myosin heavy (MHC) and light chain (LC) incorporation into myofibrils or native and synthetic thick filaments. 35S-labeled myofibrillar proteins or myosin subunits were synthesized in a reticulocyte lysate translation system after which myofibrils or myofilaments were added and incubated with these proteins in the lysate. The added filaments were then sedimented and analyzed by SDS-PAGE and fluorography to establish which of the labeled protein subunits were co-pelleted. Operationally, this co-sedimentation of labeled proteins with myofilaments has been termed 'protein incorporation'. We observed that newly synthesized MHC, LCs 1, 2 and 3 all incorporated into the thick filaments. However, the quantity and specificity of LC incorporation depended upon the structure or composition of the filaments. LCs 1 and 3 were preferentially incorporated into myofibrils and native thick filaments, whereas LC2 was selectively taken up by synthetic filaments prepared from purified myosin. These results suggest that soluble MHCs and LCs interact independently with myofilaments. This hypothesis is supported by the observation that selective removal of soluble MHCs, or of a single LC, did not alter the incorporation of the remaining myosin subunits. Similarly, MHCs synthesized in the absence of LCs also incorporated into myofilaments or myofibrils. We propose that myosin subunits are capable of independent incorporation into and exchange from myofilaments.

Size and charge heterogeneity of C-protein isoforms in avian skeletal muscle. Expression of six different isoforms in chicken muscle.

C-protein is an abundant protein, of unknown function, found in the striated muscles of all vertebrates (Offer et al., 1973). Based on differences in size, charge, antigenicity and sarcomere distribution, at least three different isoforms of this protein have been identified (Callaway & Bechtel, 1981; Yamamoto & Moos, 1983; Reinach et al., 1982; Dhoot et al., 1985). These have been termed fast-, slow- and cardiac-type isoforms, relative to their distribution in adult striated muscles. Each of these isoforms appears to be expressed sequentially during the development of the chicken pectoralis muscle (Obinata et al., 1984; Obinata, 1985). To better characterize the various isoforms of C-protein, we have reexamined its in vivo expression during avian myogenesis using a combination of 1- and 2-dimensional gel electrophoresis, cell-free translation and immunoblotting procedures. In this manuscript we demonstrate for the first time that at least four major C-protein isoforms can be distinguished in adult chicken muscles. These include a fast-type isoform in the pectoralis (PECT) muscle (Cf), a slow-type isoform in the anterior latissimus dorsi (ALD) muscle (Cs3), a second slow-type isoform in the posterior latissimus dorsi (PLD) muscle (Cs4) and a cardiac-type in the ventricle (Cc). During embryonic development of the PECT muscle two additional isoforms can be resolved. These are both slow-type isoforms based on their reactivities with ALD66, a monoclonal antibody specific for adult slow-type C-protein. These latter isoforms have been termed Cs1 and Cs2. Several of the isoforms, particularly Cs1 ands Cs3, exhibit two or more spots of different charge but identical molecular weight on 2-D gels. This observation suggests the possibility that these isoforms are post-translationally modified and possibly phosphorylated. Our data show the C-protein family in avian striated muscles to be highly complex. Additional genetic analyses and primary sequence studies will be required to distinguish transcriptional from post-transcriptional variants.

Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system.

The incorporation of newly synthesized protein into myofibrils has been examined in a cell-free system. Myofibrils were added to a reticulocyte lysate after the in vitro translation of muscle-specific poly(A)+RNA. Only a small number of the many synthesized proteins were found to associate with the exogenously added myofibrils. These proteins were all identified as sarcomeric components and had subunit mobilities (Mr) of 200, 140, 95, 86, 43, 38, 35, 25, 23, 20, and 18 kD. The association was rapid (t1/2 less than 15 min) and, for most of the proteins, relatively temperature insensitive. Except for a 43-kD polypeptide, tentatively identified as beta-actin, none of the proteins encoded by brain poly(A)+RNA associated with the myofibrils. When filaments made from purified myosin or actin were used as the "capture" substrates, only thick or thin filament proteins, respectively, were incorporated. Incorporation was substantially reduced when cross-linked myosin filaments were used. These results are compatible with a model in which proteins of the sarcomere are in kinetic equilibrium with homologous proteins in a soluble pool.

Calcium-dependent shortening of fibroblasts induced by the ionophore, A23187.

Attached, spread, chick heart fibroblasts were induced to shorten by treatment with the ionophore, A23187. The shortening resulted from the retraction of the leading lamellae and other major cell processes. The response was dependent on external calcium with a threshold close to, and a maximal effect at, physiological concentrations. The shortening was also induced in Colcemid-treated cells and therefore did not involve a depolymerization of microtubules. Indirect evidence indicates that the shortening was preceded by an increase in tension in the spread cell. The response is consistent with the effect of an increase in the cytoplasmic concentration of free calcium on a calcium-sensitive actomyosin system in the spread fibroblast. Although the retraction of non-spreading processes mimicked the intermittent retraction of similar trailing processes during normal movement of fibroblasts, the response to the ionophore differed in that the leading lamellae were also induced to retract. This difference implies that a general increase in the cytoplasmic concentration of free calcium alone cannot account for the intermittent shortening that occurs in normal movement.