INTRODUCTION

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CARDIAC HYPERTROPHY, a major risk factor for cardiac morbidity and mortality (13), is an adaptive response to a broad variety of cardiovascular stresses. Whereas multiple factors likely participate in cardiac hypertrophy, including neurohormonal factors and mutations in genes for sarcomere proteins, biomechanical forces play a prominent role (12, 21). The hypertrophic response to mechanical overload may be initially beneficial, but through incompletely described processes, cardiac hypertrophy can be maladaptive and progress to heart failure.

The Down Syndrome Critical Region-1 gene [DSCR1, also known as MCIP1 (6, 7, 9, 20, 28)] on human chromosome 21 is highly expressed in the brain, heart, and skeletal muscle and encodes a protein that interacts physically and functionally with calcineurin A, the catalytic subunit of the Ca²⁺/calmodulin-dependent protein phosphatase (PP2B) (10). Recent studies showed that overexpression of DSCR1 in hearts of transgenic mice attenuates hypertrophic responses to -adrenergic receptor stimulation or exercise training and prevents progression to dilated cardiomyopathy that otherwise results from chronic activation of calcineurin in the myocardium (19). Because calcineurin participates in signal transduction leading to cardiac hypertrophy (17), we tested the hypothesis that DSCR1 is directly induced in cardiac myocytes in response to biomechanical strain. Our results demonstrate that biomechanical strain directly induces DSCR1 gene expression, suggesting a negative feedback mechanism to regulate calcineurin activity during the hypertrophic response to biomechanical stimuli.

	METHODS

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Culture and biomechanical strain of myocytes. Neonatal rat ventricular myocytes (NRVMs) from 1-day-old Harlan Sprague-Dawley rats were isolated by previously described methods (24). Mechanical deformation was applied to a thin and transparent membrane on which cells were cultured, an approach that produces controlled biaxially uniform cellular strain as well as visualization of cells (2). After 24 h of being plated in Dulbecco's modified Eagle's medium (DMEM) containing 7% fetal bovine serum, NRVMs were washed twice with phosphate-buffered saline and made quiescent by incubating with DMEM containing 1% ITS (insulin-transferrin-sodium selenite, Sigma) supplement for ~48 h, because the signal-to-noise ratio is more prominent in quiescent cells in response to strain. To eliminate the variable of time-dependent changes due to cell age or effects of adhesion, in each experiment, all cells were cultured on the membrane for an identical time period, and cells and media from all samples were harvested at the same time. For example, in a time course experiment with strain, the time point represents the time before harvest that strain was initiated, such that the strain sample and control sample were harvested at the same time.

Animal studies. The Harvard Medical School Standing Committee on Animal Research approved the study protocol. Pressure overload was produced by transverse aortic constriction. Male FVB mice (age 8-10 wk) were anesthetized with pentobarbital (25-30 µg/g ip), and the transverse aorta was constricted by tying a 7-0 nylon suture around the vessel against a blunted 27-gauge needle with the aid of a dissecting microscope. The needle was removed, and, after recovery, hypertrophy was measured by weekly echocardiograms. In the mice used for this study, mean left ventricular mass-body weight in banded mice compared with sham mice increased by 40% at 4 wk and 64% at 12 wk; these changes are comparable to increases measured in a large cohort of mice in our laboratory followed prospectively with blinded echocardiograms (data not shown).

Northern and Western blot analyses. Northern blot analysis was performed and analyzed as previously described (24). Purified RNA (1 µg) was used for the synthesis of the 600-base pair DSCR1 cDNA probe. Western analysis was performed using a polyclonal antibody against calcineurin A (Santa Cruz, CA) (2).

Calcineurin activity assay. Calcineurin activity was assayed using a cellular calcineurin assay kit from BioMol according to the manufacturer's protocol. Phosphatase activity was measured as the dephosphorylation rate of a synthetic phosphopeptide substrate (RII peptide). The amount of PO₄ released was determined colorimetrically with the BioMol green reagents.

Promoter assays. The human DSCR1 promoter construct containing the luciferase reporter was kindly provided by Dr. R. Sanders Williams from Duke University (28). Cardiac myocytes were transfected with each reporter plasmid (15 µg) by using the calcium phosphate precipitation method (24). As an internal control for transfection efficiency, a cytomeglovirus -gal plasmid (2 µg) was cotransfected in all experiments. Cells were subjected to mechanical strain 48 h after transfection, and lysates were prepared 24 h later for luciferase and -galactosidase assays as described by the manufacturer (Applied Biosystems; Bedford, MA). The relative luciferase activity was calculated as the ratio of luciferase to -galactosidase activities and standardized to the basal activity of the unstimulated 874 to +30 DSCR1 (MCIP1) luciferase reporter construct (fold induction).

Statistics. Data are presented as means ± SE and were analyzed by Student's t-tests or ANOVA. A value of P < 0.05 was considered statistically significant.

	RESULTS

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Mechanical induction of DSCR1. Cyclic biomechanical strain (8%, 1 Hz) induced DSCR1 mRNA accumulation in a time-dependent manner in cardiac myocytes, reaching a maximum at 3 h and persisting for at least 24 h (Fig. 1, A and B). The DSCR1 mRNA level after 6 h of mechanical strain was increased 3.4 ± 0.2-fold (n = 11, P < 0.01). In addition, when NRVMs were subjected to cyclic biaxial strains of 2, 4, 8, or 12% at 1 Hz for 5 h, the induction of DSCR1 mRNA expression in NRVMs was amplitude dependent (Fig. 1C).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Time and amplitude dependence of Down Syndrome Critical Region-1 (DSCR1) mRNA induction by mechanical strain in neonatal rat ventricular myocytes (NRVMs). A: NRVMs were exposed to 0 or 8% cyclic mechanical strain (1 Hz) for various periods as indicated. Top, Northern blot analysis with 32P-labeled DSCR1 cDNA probes. Bottom, ethidium bromide-stained 28S showing equal loading of RNA. Data are representative of 4 experiments that gave nearly identical results. B: line graph showing time dependence of DSCR1 mRNA induction by mechanical strain. DSCR1 mRNA increased at 15 min, reached maximum within 3 h, and persisted for at least 24 h. Data are means ± SE of 4 independent experiments. C: amplitude dependence of DSCR1 mRNA induction by mechanical strain in NRVMs. Myocytes were exposed to 0, 2, 4, 8, and 12% cyclic mechanical strain (1 Hz) for 5 h. Top, Northern blot analysis with 32P-labeled DSCR cDNA probes. Data are representative of 3 experiments that gave similar results.

Effects of mechanical strain on the stability of DSCR1 mRNA. To determine whether mechanical strain increased DSCR1 mRNA accumulation by increasing the rate of synthesis or by decreasing the rate of degradation, NRVMs were exposed to 0% or 8% strain for 6 h and then incubated further with actinomycin D (5 µg/ml) to inhibit transcriptional activity. The half-life of DSCR1 mRNA was not affected by mechanical strain (2.98 ± 0.12 h vs. 2.87 ± 0.27 h of unstretched cells, P > 0.05, n = 3) (Fig. 2). These experiments suggest that mechanical strain increases the rate of synthesis of DSCR1 mRNA.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effects of mechanical strain on the stability of DSCR1 mRNA. NRVMs were plated on fibronectin-precoated membrane in DMEM containing 7% fetal calf serum for 24 h. After serum deprivation for 48 h, myocytes were exposed to 0 (solid circles) or 8% (open circles) cyclic mechanical strain at 1 Hz for 6 h and were further incubated with actinomycin D (5 µg/ml) for the indicated times. For each time point, total RNA (5 µg) was prepared and analyzed by Northern blotting. A: representative Northern blot showing the half-life of DSCR1 mRNA in the presence or absence of strain. B: line graph showing that the half-life of DSCR1 mRNA was not affected by strain. All values are means ± SE; n = 3. P > 0.05.

Effects of neurohormonal factors and cytokines on the induction of DSCR1 mRNA. To determine whether the effect of cyclic mechanical strain on DSCR1 mRNA expression in NRVMs is angiotensin II or endothelin dependent, NRVMs were subjected to 8% cyclic strain at 1 Hz for 6 h in the presence or absence of an AT₁ receptor or endothelin receptor antagonist. CP-191,166 (0.1 µM), an AT₁ receptor antagonist, or PD-145065 (1 µM), an endothelin receptor antagonist, had no significant effect on DSCR1 mRNA induction by cyclic mechanical strain (Fig. 3, A and B).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   A: representative autoradiograph showing the role of angiotensin II and endothelin in mechanical induction of DSCR1 mRNA. Myocytes were exposed for 6 h to 0 or 8% cyclic mechanical strain (1 Hz) in the presence or absence of CP-191,166 (0.1 µM) or PD-145065 (1 µM). Top, CP-191,166 was applied to the myocytes 30 min before mechanical strain. Bottom, ethidium bromide-stained 28S showing equal loading of RNA. B: bar graph showing the effects of angiotensin II receptor and endothelin receptor antagonists on mechanical induction of DSCR1 mRNA. All values are means ± SE (*P > 0.05, n = 3). C: representative autoradiograph showing the effects of phenylephrine (PE, 1 µM), endothelin-1 (ET, 100 nM), angiotensin II (AII, 100 nM), leukemia inhibitory factor (LIF, 1,000 U/ml), or tumor necrosis factor- (TNF-, 10 ng/ml) on mechanical induction of DSCR1 mRNA for 6 h. D: bar graph showing the effects of PE (1 µM), ET (100 nM), AII (100 nM), LIF (1,000 U/ml), or TNF- (10 ng/ml) on mechanical induction of DSCR1 mRNA. All values are means ± SE; n = 3. *P < 0.05 compared with strain.

Protein kinase C and MAP kinases in mechanical induction of DSCR1. Mechanical strain activates protein kinase C (PKC) and mitogen-activated protein (MAP) kinases in cardiac myocytes (11, 24-27). Calphostin C (1 µM), a selective PKC inhibitor (24), did not inhibit DSCR1 mRNA induction by strain (Fig. 4). In addition, neither PD-98059 (50 µM), a MAP kinase kinase inhibitor (24), nor SB-203580 (10 µM), a p38 MAP kinase inhibitor (24), inhibited the effect of mechanical strain (Fig. 4). These findings suggest that the PKC and MAP kinase pathways do not mediate mechanical induction of DSCR1 expression.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   A: representative autoradiograph showing the effects of protein kinase C and mitogen-activated protein (MAP) kinase inhibitors on induction of DSCR1 mRNA expression by mechanical strain. NRVMs were exposed for 6 h to 0 or 8% cyclic mechanical strain (1 Hz) in the presence or absence of calphostin C (CaL, 1 µM), PD-98059 (PD, 50 µM), or SB-203580 (SB, 10 µM). These inhibitors were applied to the myocytes 30 min before mechanical strain. Top, total RNA was isolated and analyzed by Northern blotting with 32P-labeled DSCR1 cDNA probes. Bottom, ethidium bromide-stained 28S showing equal loading of RNA. B: bar graph showing the effects of protein kinase C and MAP kinase inhibitors on mechanical induction of DSCR1 mRNA. All values are means ± SE; n = 3. NS, no significance from strain.

Role of Ca²+ in mechanical induction of DSCR1. Because mechanical strain increases intracellular Ca²⁺ in cardiac myocytes (1), experiments were performed to examine the effect of manipulation of intracellular Ca²⁺ concentrations on mechanical induction of DSCR1 expression. NRVMs were preincubated with the cell membrane-permeable Ca²⁺ chelator BAPTA-AM (10 µM) for 30 min and then exposed to 0 or 8% strain at 1 Hz for 6 h. Northern analysis showed that BAPTA-AM completely blocked mechanical induction of DSCR1 mRNA expression (Fig. 5 A and B). In addition, the Ca²⁺ ionophore A23187 (10 µM) induced DSCR1 mRNA expression. These data suggest that induction of DSCR1 expression by mechanical strain is mediated by intracellular Ca²⁺ signaling.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   A: representative autoradiograph showing mechanical induction of DSCR1 mRNA by a calcium-dependent mechanism. Top, Northern blot analysis of RNA extracted from cultured neonatal rat cardiomyocytes in the presence or absence of mechanical strain, A23187 (10 µM), or calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (10 µM). Bottom, ethidium bromide-stained 28S showing equal loading of RNA. Data are representative of two experiments that gave nearly identical results. B: bar graph showing mechanical induction of DSCR1 through a calcium-dependent mechanism. *P < 0.05 compared with strain; n = 3. C: representative autoradiograph showing no effect of Ca2+/calmodulin-dependent protein kinase blocker on mechanical induction of DSCR1. Top, Northern blot analysis of RNA extracted from cultured neonatal rat cardiomyocytes in the presence or absence of KN-62 (10 µM). Bottom, ethidium bromide-stained 28S showing equal loading of RNA. D: bar graph showing no effect of Ca2+/calmodulin-dependent protein kinase blocker on mechanical induction of DSCR1 mRNA. NS, no significance from strain; n = 3.

Calcineurin and induction of DSCR1. It was recently reported that overexpression of constitutively active calcineurin A upregulates DSCR1 gene expression in C2C12 myogenic cells (28). To investigate participation of calcineurin in mechanical induction of DSCR1 in cardiomyocytes, NRVMs were exposed to 8% cyclic strain at 1 Hz for 6 h in the presence or absence of cyclosporin A (1 µM), a calcineurin inhibitor. Cyclosporin A pretreatment partially attenuated mechanical induction of DSCR1 mRNA (30 ± 5%, n = 3, P < 0.01) (Fig. 6A). Cyclosporin A also blocked LIF-induced DSCR1 induction by 35 ± 4% (n = 3, P < 0.01).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   A: representative autoradiograph showing the effect of calcineurin inhibition on mechanical induction of DSCR1 mRNA. Myocytes were exposed to 0 or 8% cyclic mechanical strain (1 Hz) for 6 h in the presence or absence of cyclosporin A (CyA, 1 µM). CyA was applied to the myocytes 30 min before mechanical strain. Top, Northern blot analysis with 32P-labeled DSCR1 cDNA probes. Bottom, ethidium bromide-stained 28S showing equal loading of RNA. Data are representative of 3 experiments that gave nearly identical results. B: line graph showing mechanical strain increased calcineurin (CaN) activity. Cells were exposed to 8% cyclic mechanical strain (1 Hz) for different periods of time. Total cellular protein was extracted and CaN activity was measured using a kit. Data are means ± SE of 3 independent experiments that were performed in duplicate. *P < 0.01.

Effects of mechanical strain on DSCR1 promoter activity. Yang et al. (28) recently showed that an intragenic region between exon 3 and exon 4 of the DSCR1 gene contains a dense cluster of consensus NF-AT binding motifs (T/AGGAAANA/T/C) that are responsive to calcineurin activation. To determine whether these transcriptional regulatory elements are mechanically responsive, NRVMs were transfected with a DSCR1-luciferase reporter construct containing the consensus nuclear factor of activated T cells (NF-AT) binding motifs (874 to +30 relative to the first nucleotide of exon 4). As showed in Fig. 7, the reporter activity was stimulated 2.3-fold by mechanical strain, and this was abolished by cyclosporin A (1 µM) pretreatment, indicting that mechanical stimulation of the DSCR1 promoter is mediated by activation of calcineurin.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Role of CaN-NF-AT in induction of DSCR1 promoter activity by mechanical strain. Cardiac myocytes were transfected with DSCR1 promoter-reporter plasmid (15 µg) and cytomeglovirus -gal plasmid (2 µg). Forty eight hours after transfection, cells were subject to 0 or 8% mechanical strain on the absence or presence of CyA (1 µM), and lysates were prepared 24 h later for luciferase activity assays and -galactosidase activity assays. Relative luciferase activity was calculated as the ratio of luciferase to -galactosidase activity and expressed as fold activation to the basal activity of DSCR1-luciferase reporter construct. Data are means ± SE of 3 independent experiments that were performed in triplicate.

Induction of DSCR1 in pressure-overload hypertrophy. To determine whether DSCR1 is mechanically induced in vivo, we studied the effect of pressure overload on DSCR1 mRNA induction in a mouse model of pressure-overload hypertrophy induced by transverse aortic constriction. DSCR1 mRNA was markedly induced in the left ventricles of banded mice compared with those of sham-operated mice at both 4 and 12 wk after surgical operations (Fig. 8).

View larger version (36K): [in this window] [in a new window]   Fig. 8.   DSCR1 mRNA is induced in pressure-overloaded left ventricles. Northern blot analysis demonstrated that DSCR1 mRNA levels were increased in the hearts of banded mice (B) compared with those of sham-operated mice (S).

	DISCUSSION

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These data demonstrate that biomechanical strain directly induces the DSCR1 gene, which encodes a calcineurin inhibitory protein in cardiac myocytes. The induction of DSCR1 mRNA by mechanical strain was time and amplitude dependent. In addition, mechanical induction of DSCR1 mRNA was angiotensin II and endothelin independent and was partially blocked by calcineurin inhibition. To our knowledge, this is the first report that mechanical strain directly regulates calcineurin activity and its inhibitory protein DSCR1 in cardiac myocytes. The induction of DSCR1 mRNA expression by cyclic mechanical strain in cardiac myocytes was rapid and highly reproducible. Increased DSCR1 mRNA was detected within 15 min after mechanical stimulation, suggesting a rapid feedback mechanism for regulating the calcineurin system. Yang and colleagues (28) recently reported that the DSCR1 gene is responsive to calcineurin signaling, whereas a second calcineurin inhibitor (MCIP2) was not calcineurin responsive. Our results show that mechanical induction of DSCR1 mRNA is intracellular Ca²⁺ dependent and partially dependent on calcineurin signaling, suggesting that other Ca²⁺-dependent mechanisms may also play a role in the regulation of DSCR1 expression.

DSCR1 mRNA is upregulated in a mouse model of pressure overload-induced hypertrophy. The induction of DSCR1 mRNA by pressure overload was persistent for at least 12 wk after aortic banding. This raises an interesting issue of what responses to mechanical overload are sustained versus transient. It has been reported that pressure overload increases calcineurin activity in the heart. The increase of calcineurin activity can be detected as early as 30 min and persists for at least 3 wk (14, 22, 30). Therefore, we speculate that the sustained increase of DSCR1 mRNA expression is an adaptive response to a persistent increase of calcineurin activity caused by pressure overload. In addition, pressure overload activates neurohumoral factors that upregulate DSCR1 gene expression in cultured cardiac myocytes; these factors may play a role in the sustained induction of DSCR1 mRNA expression. It would be useful to show that the DSCR1 protein expression increases in response to pressure overload. However, there is currently no antibody widely available to study protein expression.

Calcineurin is a serine-threonine protein phosphatase that participates in a wide variety of biological responses, including lymphocyte activation, neuronal and muscle development, axonal pathfinding, and morphogenesis of vertebrate heart valves (3). Calcineurin may be subjected to negative regulation by proteins that reduce its ability to dephosphorylate substrates such as NF-ATc family members, thereby preventing their nuclear localization (3). Recent studies have implicated a role for calcineurin-dependent signaling pathways in cardiac hypertrophy. Molkentin and associates (17) reported that overexpression of a truncated constitutively active calcineurin A induces cardiac hypertrophy. Furthermore, cyclosporin A prevents the development of cardiac hypertrophy in response to some, but apparently not all, hypertrophic stimuli (5, 14, 23). Recently, Rothermel et al. (19) reported that forced overexpression of DSCR1 under control of the cardiac specific, -myosin heavy chain promoter attenuated cardiac hypertrophy induced by cardiac specific expression of a constitutively active form of calcineurin, -adrenergic receptor stimulation, or exercise training. The present study supports the concept that endogenous negative feedback regulation of calcineurin activity may occur both in vitro and in vivo. This raises the intriguing possibility that one reason that several studies have reached different conclusions regarding the role of calcineurin role in cardiac hypertrophy is different degrees of negative feedback by endogenous inhibitors such as DSCR1. It is important to recognize that other unknown mechanisms of calcineurin inhibition or inactivation may also play a role in the reduction of calcineurin activity.

In preliminary studies in our laboratory with DNA microarrays with the conditions used in this study, DSCR1 is among a very small subset of biomechanically induced genes in cardiac myocytes (~5 genes per 1,000 increased at least twofold). This suggests that induction of DSCR1 mRNA is part of a restricted molecular response to biomechanical stimuli, similar to our prior observations in vascular smooth muscle cells (4). Further studies of the mechanisms controlling DSCR1 gene induction by mechanical strain may lead to new understanding of cardiac hypertrophy.