In a rat model of long-lasting pressure-overload hypertrophy, we investigated whether changes in the relative expression of myocardial actin isoforms are among the early signs of ventricular mechanical dysfunction before the transition toward decompensation. Forty-four rats with infrarenal aortic banding (AC rats) were studied. Hemodynamic parameters were measured 1 mo (AC1 group; n = 20) or 2 mo (AC2; n = 24) after aortic ligature. Then subgroups of AC1 and AC2 left ventricles (LV) were used to evaluate 1) LV anatomy and fibrosis (morphometry), 2) expression levels (immunoblotting) and spatial distribution (immunohistochemistry) of alpha-skeletal actin (α-SKA), alpha-cardiac actin (α-CA), and alpha-smooth muscle actin (α-SMA), and 3) cell mechanics and calcium transients in enzimatically isolated myocytes. Although the two AC groups exhibited a comparable degree of hypertrophy (+30% in LV mass; +20% in myocyte surface) and a similar increase in the amount of fibrosis compared with control animals (C group; n = 22), a worsening of LV mechanical performance was observed only in AC2 rats at both organ and cellular levels. Conversely, AC1 rats exhibited enhanced LV contractility and preserved cellular contractile behavior associated with increased calcium transients. Alpha-SKA expression was upregulated (+60%) in AC1. In AC2 ventricles, prolonged hypertension also induced a significant increase in α-SMA expression, mainly at the level of arterial vessels. No significant differences among groups were observed in α-CA expression. Our findings suggest that α-SKA expression regulation and wall remodeling of coronary arterioles participate in the development of impaired kinetics of contraction and relaxation in prolonged hypertension before the occurrence of marked histopathologic changes.
- aortic ligature
cardiac hypertrophy in response to pressure overload is initially beneficial but eventually leads to heart failure (HF), a major cause of morbidity and mortality in the Western countries. In the hypertrophied heart, ventricular remodeling is mainly characterized by fibrosis, insufficient vascularization, and alterations in cardiomyocytes, including changes in number, shape, and gene expression. The abnormalities in the ventricular myocardial tissue accelerate the development of diastolic and systolic dysfunction, potentially resulting in HF (2, 5, 10, 17). The fundamental mechanisms responsible for the transition from compensated to decompensated cardiac hypertrophy are only partially defined (1, 31). Many studies point to death of cardiomyocytes and changes in the cellular/molecular mechanisms regulating extracellular matrix composition and organization as the most relevant factors in the transition from compensatory hypertrophy to pump failure in experimental and human hypertension (1, 10, 12, 19, 23, 30, 34, 40, 42). It has been also suggested that the accumulation of cytoskeletal proteins, tubulin and desmin, probably counteracting the increased strain on the myocardium due to chronic pressure overload, as well as changes in intercalated disc proteins may lead to cytoskeletal stiffness and reduced intercellular communication resulting in contractile dysfunction before the transition to overt HF (18, 31).
Although abundant evidence suggests that isoform shifts in thick- and thin-filament proteins as well as myofilament protein phosphorylation contribute substantially to the contractile deficit of cardiac failure (7, 18, 22, 33, 37, 39), the contribution of changes in cardiac actin isoform expression to the transition from compensatory to early maladaptive processes in the hypertrophic heart disease has been less explored. We have recently reported (36) that the expression of alpha-skeletal actin is upregulated since the initial stages of compensated pressure-overload hypertrophy increases proportionally to the degree of ventricular hypertrophic response to pressure overload and leads to a parallel increase in myocardial contractility, playing a relevant role in preserving cardiac mechanical performance. In the present study, we tested the hypothesis that changes in the relative expression of myocardial actin isoforms are among the early signs of ventricular mechanical impairment during long-lasting pressure overload, before the appearance of marked histopathological alterations of the LV myocardium. For this purpose, we measured cardiac hermodynamics in intact animals, cellular contractile performance and calcium transients in isolated ventricular myocytes, and expression levels of alpha-skeletal (α-SKA), alpha cardiac (α-CA), and α-smooth muscle-actin (α-SMA) in the LV of aortic banded rats with short-lasting or long-lasting pressure overload.
The study population consisted of 66 male 4-mo-old Wistar rats (Rattus Norvegicus), individually housed in Plexiglas cages from the beginning of the experimental protocol in a temperature controlled room (22–24°C), with light on between 7 AM and 7 PM. The experimental protocol was approved by the Veterinary Animal Care and Use Committee of the University of Parma and conforms with the APS's Guiding Principles in the Care and Use of Animals and the National ethical guidelines (Italian Ministry of Health; D.L.vo 116, January 27, 1992).
Forty-four animals were submitted to infrarenal abdominal aortic coarctation to induce left ventricular hypertrophy (AC group), whereas 22 animals were used as control group (C group). In AC group, invasive hemodynamic measurements were performed 1 mo (n = 20; AC1 group) or 2 mo (n = 24; AC2 group) after surgery. Then, the hearts of AC and C rats were subdivided in different subgroups, respectively submitted to 1) morphometrical and immunohistochemical analyses (11 AC1, 14 AC2, and 10 C rats) to define left ventricular geometry, amount of perivascular and interstitial fibrosis in the left ventricular wall, and spatial distribution of α-SKA and α-SMA in the left ventricular myocardium (subgroups of 5 AC1, 5 AC2, and 5 C rats); 2) electrophoretic and immunoblot analysis (4 AC1, 5 AC2, 5 C rats) to quantitatively evaluate α-SKA, α-CA, and α-SMA expression; and 3) enzymatic digestion (5 AC1, 5 AC2, 7 C rats) to obtain isolated ventricular myocytes for measuring cell mechanics and calcium transients and determining cell size and the percentage of mononucleated and binucleated cells.
Surgery: Aortic Coarctation
Rats were anaesthetized with Tiletamine chloride + zolazepam chloride (Zoletil-100, Virbac-France; 0.3 ml/kg im). The abdominal aorta was dissected free, and a silk ligature of the vessel was performed between the two renal arteries (caliber of coarctation: 0.4 mm, corresponding to a lumen reduction of ∼80%). In C rats, the ligature was not tied. After surgery, all animals received antibiotic therapy with gentamicine sulfate for 3 days (Aagent, Fatro, Milan, Italy; 0.2 ml/kg im).
24) (software package CHART B4.2).1) LV systolic pressure (LVSP), 2) LV end-diastolic pressure (LVEDP) measured immediately before the rapid upstroke in the LV pressure tracing, 3) the peak rate of rise and decline of LV pressure (±dP/dt), taken as indexes of myocardial contractility, 4) isovolumic contraction time (IVCT: duration of isovolumic contraction), and 5) the time constant of isovolumic LV relaxation (TAU), computed from −dP/dt to 5 mmHg above LVEDP (
Morphometric Measurements and Immunohistochemistry
In 11 AC1, 14 AC2, and 10 C anaesthetized with Zoletil-100+Domitor, the heart was arrested in diastole by injecting 2 ml of cadmium chloride solution (100 mM iv) and excised. The right ventricle and the LV, including the septum, were separately weighted and fixed in 10% buffered formalin solution. In all animals, the weights of liver and lungs and the length of the tibia were also determined as additional indexes of compensation or decompensation.
The volume of the LV myocardium was computed by dividing ventricular weight by the specific gravity of the tissue (1.06 g/ml). LV chamber length was measured from the apex to the aortic valve. The equatorial transverse section of the LV (1 mm thick), cut perpendicularly to the major axis, allowed the morphometric computation (Image Pro-plus, Media Cybernetics, Bethesda, MD) of the LV equatorial diameter. The LV chamber volume was then calculated according to the Dodge equation, which equalizes the ventricular cavity to an ellipsoid (9). The section was finally embedded in paraffin.
Five-micrometer-thick serial sections were obtained from the equatorial slice and used for morphometrical and immunohistochemical analyses. One section, stained with blue aniline Masson's trichrome, was analyzed by optical microscopy (magnification ×250) to evaluate the volume fraction of perivascular and interstitial fibrosis in the LV myocardium. According to a procedure previously described (36), for each section a quantitative evaluation of the fibrotic tissue was performed in 60 consecutive fields from subendocardium, midmyocardium, and subepicardium with the aid of a grid defining a tissue area of 0.160 mm2 and containing 42 sampling points, each covering an area of 0.0038 mm2. To define the volume fraction of fibrosis, the number of points overlying myocardial fibrosis was counted and expressed as percentage of the total number of points explored. In five animals of each group, other sections were used for immunohistochemistry with anti-α-SKA antibody (7) diluted at 1:100 in Tris-buffered saline (TBS-10 mM Tris, 154 mM NaCl, pH 7.4) and anti-α-SMA (1:10) (35). Immunoperoxidase staining was performed essentially as previously described (32). After being stained, sections were observed using a photomicroscope (Zeiss, Oberkochen, Germany). Images were acquired with a high sensibility color camera (JVC, DCS) and subsequently analyzed using the software Image-Pro-plus. Myocardial areas were considered positive to the immunostaining when their pixel intensity values overcame background values. Positive areas were then expressed as percentage of the total myocardial area explored.
Electrophoretic and Immunoblot Analysis
The hearts of 4 AC1, 5 AC2, and 5 C were arrested in diastole as described above and excised. The right and left ventricles were separately weighted and frozen at −80°C. The LV tissue was mechanically fragmented in liquid nitrogen, homogenized in sample buffer (62.5 mM Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue), and boiled for 5 min. For each animal, 3 μg (α-SKA and α-CA) or 20 μg (α-SMA) of proteins were separated on 10% polyacrylamide gels (20) and electroblotted on nitrocellulose membranes (Protran, Schleicher & Schuell, Dassel, Germany) according to Towbin et al. (38). Membranes were incubated for 2 h at room temperature with anti α-SKA (α-SKA1–1:500), anti α-CA (1:5,000) (8), and anti-α-SMA (1:500) (35) antibodies, diluted in TBS solution containing 3% bovine serum albumin (BSA) and 0.1% Triton X-100. After three washes with TBS containing 0.1% Triton X-100, a second incubation was performed for 1 h at room temperature with peroxidase-conjugated affinity purified goat anti-rabbit or anti-mouse antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) at a dilution of 1:10,000 in TBS containing 0.1% BSA and 0.1% Triton X-100. Peroxidase activity was developed using the ECL Western blotting system (Amersham, Rahn, Zürich, Switzerland), according to the instructions of the manufacturer. To determine the expression of actin isoforms, blots were scanned (Arcus II; Agfa, Mortsel, Belgium), and the intensity of the band was quantified by means of the ImageQuant Program (Image Quant Analysis, Molecular Dynamics, Sunnyvale, CA).
From the hearts of 5 AC1, 5 AC2, and 7 C, individual ventricular myocytes were enzymatically isolated by collagenase perfusion in accordance with a procedure previously described (43). Briefly, the rat heart was removed and rapidly perfused at 37°C by means of an aortic cannula with the following sequence of solutions: 1) a calcium-free solution for 5 min to remove the blood [calcium-free solution contained the following (in mM): 126 NaCl, 22 dextrose, 5.0 MgCl2, 4.4 KCl, 20 taurine, 5 creatine, 5 Na pyruvate, 1 NaH2PO4, and 24 HEPES (pH = 7.4, adjusted with NaOH)], and the solution was gassed with 100% O2; 2) a low-calcium solution (0.1 mM) plus 1 mg/ml type 2 collagenase (Worthington Biochemical), and 0.1 mg/ml type XIV protease (Sigma, Milan, Italy) for about 20 min; and 3) an enzyme-free, low-calcium solution for 5 min. The LV was then minced and shaken for 10 min. The cells were filtered through a nylon mesh and re-suspended in low-calcium solutions: 0.1 mM (for 30 min) and 0.5 mM (additional 30 min). Then, cells were used for measuring sarcomere shortening and calcium transients.
Smears were also made, and LV cells were stained with propidium iodide. For each group, ∼500 cells were analyzed by optical microscopy to calculate cell surface area and the relative fraction of mononucleated and binucleated LV cardiomyocytes.
LV myocytes were placed in a chamber mounted on the stage of an inverted microscope (Nikon-Eclipse TE2000-U, Nikon Instruments, Florence, Italy) and superfused (∼1 ml/min at 37°C) with a Tyrode solution containing (in mM): 140 NaCl, 5.4 KCl, 1 MgCl2, 5 HEPES, 5.5 glucose, and 1.8 CaCl21, and 40 AC2), the following parameters were computed to assess cellular mechanical properties: mean diastolic sarcomere length, time to 10% shortening (T10%S), fraction of shortening (FS), time-to-peak shortening (TPS), maximal rates of shortening and re-lengthening (±dL/dt), and time to 90% re-lengthening (T90%R). Steady-state contraction of myocytes was achieved before data recording.
In a subgroup of 17 C, 20 AC1, and 18 AC2 cells, Ca2+ transients were measured simultaneously with mechanical properties. Ca2+ transients were determined by epifluorescence after loading the myocytes with 10 μM fluo 3-AM (Invitrogen, Carlsbad, CA) for 30 min. Excitation length was 480 nm, with emission collected at 535 nm using a ×40 oil objective. Fluo 3 signals were expressed as normalized fluorescence (F/F0: fold increase). The time course of the fluorescence signal decay was described by a single exponential equation, and the time constant (tau) was used as a measure of the rate of intracellular calcium clearing.
The SPSS statistical package was used (SPSS, Chicago, IL). Normal distribution of variables was checked by means of the Kolmogorov-Smirnov test. Statistics of variables included mean ± SD, one-way ANOVA (post hoc analysis: Games-Howell test). Statistical significance was set at P < 0.05.
Hemodynamics (22 C, 20 AC1, and 24 AC2 Rats)
The heart rate measured under anesthesia was similar in the three groups (215 ± 20, 224 ± 36, and 212 ± 18 ms in C, AC1, and AC2, respectively). Compared with C animals, LVSP was significantly higher in AC1 and AC2 groups (P < 0.01), although the increment was larger in AC1 (P < 0.05 AC1 vs. AC2; Fig. 1A). LVEDP increased only in AC2 rats (P < 0.05 vs. C and AC1; Fig. 1B). In AC1 group, the rate of ventricular pressure rise (+dP/dt) significantly increased (P < 0.01 vs. C), resulting in a significant decrease of the total IVCT compared with both C and AC2 (P < 0.01 vs. C, and P < 0.05 vs. AC2; Fig. 1, C and D). The rate of LV pressure decline (−dP/dt) was also higher in AC1 rats (P < 0.01 vs. C, and P < 0.05 vs. AC2; Fig. 1E).
Cell Mechanics and Calcium Transients
Globally, 132 cells were used for cell mechanics (50 in C, 42 in AC1, and 40 in AC2). In a subgroup of 55 cells (17 C, 20 AC1, and 18 AC2), calcium transients were also simultaneously recorded. Figure 2A shows recordings from representative ventricular myocytes isolated from C, AC1, and AC2 hearts. The average diastolic sarcomere length was comparable in all groups (average sarcomere length equal to 1.724 ± 0.2 μm in C cells, 1.720 ± 0.1 in AC1, and 1.720 ± 0.2 in AC2). In AC1, cardiomyocyte contractile performance was preserved, whereas a worsening of cell mechanics was observed in AC2 cells compared with C. Specifically, AC1 myocytes exhibited average values of most parameters similar to those measured in C cells (Fig. 2, B–F) associated with an increased rate of relaxation as indicated by the 13% reduction in the time to 90% re-lengthening (P < 0.05 vs. both C and AC2; Fig. 2G). Conversely, in AC2 ventricular myocytes, the sarcomere fraction of shortening as well as the maximal rate of shortening were significantly reduced (approximately −20%, P < 0.05; Fig. 2, C and E). In addition, the time to peak shortening (TPS) was higher than in AC1 (P < 0.05; Fig. 2D).
To determine whether changes in intracellular calcium cycling contributed to altering myocyte mechanical properties, calcium transients were evaluated together with cell shortening in the three groups of myocytes. The impaired cell contraction in AC2 cells (Fig. 3A) was associated with a calcium cycling comparable to that recorded in C (Fig. 3, A–C). Conversely, in AC1, the normal pattern of cell contraction (Fig. 3A) was accompanied by a significant increase in the calcium transient amplitude (P < 0.01 vs. C, and P < 0.05 vs. AC2; Fig. 3B). No significant differences among groups were observed in the fluorescence signal decay (Fig. 3C).
Organ Weights, Cardiac Anatomy, and Tissue Morphometry (10 C, 11 AC1, and 14 AC2 Rats)
The weights of liver and lungs as well as the tibial length exhibited comparable values in all groups of animals (Table 1), indicating the absence of extra-cardiac anatomical signs of decompensation in the two AC groups.
AC1 and AC2 animals exhibited a similar hypertrophic response of the myocardium to pressure overload as suggested by the comparable increases in LV weight normalized to body weight (approximately +30% compared with C rats) (P < 0.01; Table 1). Conversely, LV chamber remodeling was markedly affected by the duration of pressure overload. Chamber volume was significantly larger in AC2 compared with both C (P < 0.01) and AC1 (P < 0.05; Table 1). In addition, the LV mass-to-chamber volume ratio was significantly reduced in AC2 hearts (P < 0.01 vs. C; Table 1), indicating an imbalance between chamber dilation and LV-mass increase.
Negligible values of perivascular and interstitial fibrosis, including small foci of collagen accumulation uniformly distributed throughout the LV wall, were observed in C hearts (Fig. 4, A and B). In AC groups, the total amount of fibrosis was significantly higher than in C due to an increase in both perivascular and interstitial fibrosis (P < 0.01 vs. C; Fig. 4, A and B). No significant difference between the two AC groups was detected in both the size and the geometrical properties of the myocardial damage. The volume fraction of myocytes was only slightly reduced (∼3%) in AC1 and AC2 ventricles (Fig. 4C).
Cell Size and Mononucleated-to-Binucleated Cardiomyocyte Ratio (490 C, 500 AC1, and 475 AC2 Cells)
The surface area of AC1 and AC2 ventricular myocytes increased by ∼20% compared with C (P < 0.05; Fig. 4D). The proportion between binucleated and mononucleated ventricular myocytes did not reveal any significant differences among groups (average percentages of binucleated and mononucleated cells reaching ∼90% and 10%, respectively).
Alpha-Actin Isoform Expression (Immunohistochemistry: 5 C, 5 AC1, and 5 AC2; Immunoblotting: 5 C, 4 AC1, and 5 AC2)
In accordance with data previously reported (7, 36), AC1 LVs exhibited significantly higher values of α-SKA expression than C (average increase ∼60%; P < 0.01) and AC2 hearts (P < 0.05) (Fig. 5, A and B), with α-SKA distribution in myocardial fibers preferentially located in the middle and subendocardial ventricular layers as well as in cardiomyocytes surrounding areas of collagen accumulation. Conversely, the increase in the LV mass in AC2 group was not associated with changes in α-SKA expression (Fig. 5, A and B). In addition, AC2 group constantly showed a homogeneous spatial distribution of α-SKA throughout the left ventricular wall, resulting in only negligible differences compared with C group (Fig. 5D). No significant differences among groups were observed in α-CA expression (Fig. 5C).
In all groups, α-SMA staining was mostly located in the media of the vessel wall. In LV myocardium of both AC groups, α-SMA-positive myofibroblasts were rarely found in interstitial fibrosis and were slightly more abundant in periarterial fibrosis (Fig. 6B, c–f). However, as assessed by Western blotting, α-SMA expression levels were significantly higher in AC2 compared with both C and AC1 (P < 0.01; Fig. 6A, A and B).
The present study supports the hypothesis that specific changes in the relative expression of α-actin isoforms, occurring during long-lasting pressure overload, contribute to the appearance of early signs of impaired ventricular mechanical function before the occurrence of severe myocardial structural damage and to the transition toward decompensation. The current investigation demonstrates that changes in mechanical performance take place early and are more evident at the cellular level than at the organ level, suggesting that the myocyte compartment participates importantly in the cardiac dysfunction that develops with the progression of the hypertrophic disease.
In short-lasting pressure overload (AC1 group), the parallel increase in α-SKA expression and ventricular contractile efficiency confirms previous findings from our group and other investigators (6, 14, 36). Alpha-SKA upregulation and the specific focal distribution of the protein in the hypertrophied ventricular myocardium may represent an attempt to compensate cardiac function, providing mechanical advantages that help to normalize cardiac hemodynamics in the face of increased load. It characterizes the initial adaptation to an acute cardiac overload, when the organ is submitted to the greatest imbalance between increased demand and functional capacity, and the mechanisms responsible for changes in the myocardial phenotype are most active (28).
Although the prolongation of hypertension did not result in increased degree of hypertrophy nor in larger extracellular matrix alterations in AC2 compared with AC1 animals, a mild deterioration of LV mechanical performance occurred, as indicated by the increase in end-diastolic ventricular pressure, the decrease in systolic ventricular pressure, and the lower values of ±dP/dt. The initial signs of impaired ventricular function were associated with 1) loss of the positive linear relationship between increase in myocardial mass and levels of α-SKA expression, and 2) changes in the spatial distribution of the protein that appeared uniformly expressed in the ventricular myocardium, as in C hearts. The mild accumulation of extracellular matrix, similar in size to that observed in AC1, should not contribute in reducing cardiac mechanical performance in AC2 hearts. Similar considerations apply to the remodeling of LV chamber geometry: despite initial evidence of an increase in LV chamber volume, not proportional to the growth of the entire LV mass, no thinning in wall thickness was observed in AC2 compared with AC1 (3). Finally, our data do not support a global depression of actin isoform synthesis in AC2 cardiomyocytes, as indicated by the preserved expression levels of α-CA, but rather a specific inability to maintain an elevated α-SKA production.
Data obtained at the cellular level support the hypothesis that the cellular compartment of myocardial tissue might play the most relevant role in triggering contractile dysfunction before the occurrence of marked extracellular matrix alterations and myocyte loss. Indeed, in the absence of blood pressure and neuronal/hormonal modulation as well as extracellular matrix and adjacent myocyte influences that affect and can support heart function “in vivo,” AC2 unloaded ventricular myocytes exhibited a decline in the contractile behavior more evident than at the organ level, mainly involving cell shortening. Specifically, AC2 cells exhibited a significant reduction in both fraction of shortening and contraction velocity compared with C and AC1. In the absence of changes in the time course of calcium transients, the altered cell kinetics in AC2 should be mainly attributable to changes in the calcium-myofilament interaction and/or intrinsic cell mechanical properties (e.g., isoform shift in myofilament proteins leading to a reduced efficiency of myofilament interaction). Conversely, AC1 isolated cardiomyocytes showed normal cell contractility and increased relaxation velocity, associated with an increased amplitude of calcium transient. An increased calcium transient induced by different mechanisms (such as greater Ca2+ influx during a longer action potential, increased sensitivity to Ca2+-releasing agonists in RyR) constitutes a common finding during early developing of pressure-overload cardiac hypertrophy and is considered an early compensatory mechanism to meet the increased work load (4). The higher peak of intracellular Ca2+ concentration would also favor SR Ca2+ uptake during relaxation as a result of a substrate concentration-dependent increase in enzyme velocity. Accordingly, a higher relaxation velocity was observed in AC1 cardiomyocytes. Although we did not see evident differences among groups in the temporal relationship between calcium transient and development of cell contraction, an increased Ca2+ transient in the absence of increased fraction of shortening suggests a reduced myofilament calcium sensitivity in AC1 myocytes. It is conceivable that the increased sarcoplasmic reticulum Ca2+ release constitutes a compensatory mechanism to maintain a normal fraction of shortening despite the reduced myofilament calcium sensitivity. This could also explain the behavior of AC2 cells where, in the absence of a significant increase in calcium transient, cell shortening was reduced. The increase in α-SKA expression in AC1 can contribute to the ability of the cell to generate force when mechanically loaded without affecting cell shortening (25). This interpretation is in accordance with the in vivo hemodynamic data obtained in AC1 group in which significantly higher values of +dP/dt were measured, indicating a higher rate of ventricular pressure rise during isometric contraction. As a whole, our data suggest that, in addition to actin isoform shift, early changes in calcium homeostasis evoked by pressure overload contribute to preserve cellular mechanical performance and constitute early adaptive adjustments of myocardium to cope with the circulatory overload. We can also exclude that differences in cellular mechanical properties between the two AC groups result from changes in the cellular composition of LV myocardium. Indeed, besides a similar degree of cardiomyocyte hypertrophy, we did not find any differences among groups in both the proportion between mononucleated and binucleated cells and cell size heterogeneity, which have been shown to markedly affect cell function (29).
Alpha-SMA staining was essentially detected in the media of the vessel wall in all groups. In AC2 animals, the expression of this protein was significantly higher than both C and AC1. In accordance with recent data obtained in mice with interrenal aortic banding (11, 15), the marked increase in SMA expression in long-lasting pressure overload could be mainly attributed to the proliferation/hypertrophy of vascular smooth muscle cells in the media, typical hypertension-related change in resistant vessels including the coronary arteries. Alpha-SMA-positive cells in the adventitia and perivascular regions, attributable to differentiation of fibroblasts into myofibroblasts (16), were negligible in both AC groups. Activation of myofibroblasts has been described in experimental pressure-overload hypertrophy (19) and is well known to play an important role in vascular remodeling leading to increased deposition of extracellular matrix after human and experimental myocardial infarct (7, 41). This phenomenon, however, was practically absent in our models of hypertension and is in accordance with the limited size of fibrosis observed in both AC groups.
In conclusion, our results are consistent with previous work showing that, before the transition to overt ventricular dysfunction, early signs of maladaptation mainly occurs in the cellular compartment of myocardial tissue as a multi-factorial process involving changes in different protein expression and in intracellular signaling pathways.
A better understanding of early adjustments to overload and the underlying cellular mechanisms should add more insight on the transition from adaptative to maladaptative responses to prolonged hemodynamic perturbation. In this study, we show that the inability of cardiomyocytes to maintain an elevated α-SKA expression in response to a long-lasting hemodynamic stress and changes in intracellular calcium cycling participate in the multi-factorial process triggering the transition from compensated hypertrophy toward LV mechanical dysfunction. Our results do not exclude the contribution of additional factors to the development of maladaptive responses to the prolonged work load, including changes in sarcomeric, regulatory, or cytoskeletal proteins, as previously reported (2, 4, 6, 13, 18, 21, 26, 27, 31). However, our findings suggest that manipulation of actin isoform expression may represent an additional useful tool to better understand and possibly influence the transition from compensatory to maladaptive processes in pressure-overload hypertrophy.
This work was supported by grants from the Italian Ministry of Education, University and Research (MIUR-PRIN 2005), the Italian National Cardiovascular Research Institute, and Swiss National Science Foundation (3100A0-109879 to C. Chaponnier and PMPDA-102408 to S. Clement).
We gratefully acknowledge Giuseppe Celetta and Anita Hiltbrunner for invaluable technical help.
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