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To examine the contribution of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) to early heart failure, we subjected transgenic (TG) mice expressing SERCA2a gene and wild-type (WT) mice to aortic stenosis (AS) for 7 weeks. At an early stage of hypertrophy (4-week AS), in vivo hemodynamic and echocardiographic indices were similar in TG and WT mice. By 7 weeks of AS, which is the stage of early failure in this model, TG mice with AS had lower mortality than WT mice with AS (6.7% versus 29%). The magnitude of left ventricular (LV) hypertrophy was similar in WT and TG 7-week AS mice. In vivo LV systolic function was higher in TG than in WT 7-week AS mice. In LV myocytes loaded with fluo-3, fractional cell shortening and the amplitude of the [Ca(2+)](i) transients were higher in TG than in WT 7-week AS mice under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca(2+)](o), 25 degrees C). The rates of relengthening and decay in [Ca(2+)](i) were faster in TG than in WT 7-week AS myocytes. In myocytes from WT 7-week AS compared with sham-operated WT mice, contractile reserve in response to rapid pacing was depressed with impaired augmentation of both peak-systolic [Ca(2+)](i) and the SR Ca(2+) load. In contrast, contractile reserve and the capacity to augment SR Ca(2+) load were maintained in TG 7-week AS mice. SERCA2a protein levels were depressed in WT 7-week AS mice, but were preserved in TG 7-week AS mice. These data suggest that defective SR Ca(2+) loading contributes to the onset of contractile failure in animals with chronic pressure overload.

Cyclin-dependent kinase 2 (cdk2) plays a critical role in the G1- to S-phase checkpoint of the cell cycle. Adult cardiomyocytes are believed to withdraw from the cell cycle. To determine whether forced overexpression of cdk2 results in altered cell-cycle regulation in the adult heart, we generated transgenic mice specifically overexpressing cdk2 in hearts. Transgenic hearts expressed high levels of both cdk2 mRNA and catalytically active cdk2 proteins. Cdk2 overexpression significantly increased the levels of cdk4 and cyclins A, D3, and E. There was an increase in both DNA synthesis and proliferating cell nuclear antigen levels in the adult transgenic hearts. The ratio of heart weight to body weight in cdk2 transgenic mice was significantly increased in neonatal day 2 but not in adults compared with that of wild-type mice. Analysis of dispersed individual adult cardiomyocytes showed a 5.6-fold increase in the proportion of smaller mononuclear cardiomyocytes in the transgenic mice. Echocardiography revealed that transgenic heart was functionally normal. However, adult transgenic ventricles expressed beta-myosin heavy chain and atrial natriuretic factor. Surgically induced pressure overload caused an exaggerated maladaptive hypertrophic response in transgenic mice but did not change the proportion of mononuclear cardiomyocytes. The data suggest that overexpression of cdk2 promotes smaller, less-differentiated mononuclear cardiomyocytes in adult hearts that respond in an exaggerated manner to pressure overload.

Background: Neuregulins are a family of peptide growth factors that promote cell growth and viability. The potential role of neuregulin-erbB signaling in hypertrophic growth and later failure in the adult heart in vivo is not known., Methods and Results: We used ribonuclease protection assays to quantify mRNA levels of neuregulin, erbB2, and erbB4 in left ventricular (LV) tissue and myocytes of normal rats and rats with aortic stenosis with pressure-overload hypertrophy 6 and 22 weeks after banding. At both stages of hypertrophy, Northern blot analyses of mRNA from LV myocytes showed upregulation of atrial natriuretic peptide, a molecular marker of hypertrophy (P<0.05). LV tissue neuregulin message levels were similar in animals with aortic stenosis compared with controls (P=NS) and were not detectable in myocytes. LV erbB2 and erbB4 message levels in LV tissue and myocytes were maintained during early compensatory hypertrophy in 6-week aortic stenosis animals compared with age-matched controls; in contrast, erbB2 and erbB4 message levels were depressed in 22-week aortic stenosis animals at the stage of early failure (both P<0.01 vs age-matched controls). Immunoblotting of erbB2 and erbB4 also showed normal protein levels in 6-week aortic stenosis animals compared with controls; however, erbB2 and erbB4 protein levels were depressed in 22-week aortic stenosis animals (48% decrease in erbB2, P<0.05, and 43% decrease in erbB4, P<0.01) relative to age-matched controls., Conclusions: The neuregulin receptors erbB2 and erbB4 are downregulated at both the message and protein levels at the stage of early failure in animals with chronic hypertrophy secondary to aortic stenosis. These data suggest a role for disabled erbB receptor signaling in the transition from compensatory hypertrophy to failure.

Objectives: The objective of this study was to examine gender differences in left ventricular (LV) function and expression of cardiac genes in response to LV pressure overload due to ascending aortic stenosis in rats., Background: Clinical studies have documented gender differences in the pattern of adaptive LV hypertrophy. Whether these differences result from intrinsic differences in molecular adaptation to pressure overload between men and women, or are related to other factors is not known., Methods: Male (n = 8) and female (n = 8) Wistar rats underwent ascending aortic stenosis and were studied 6 weeks after banding with gender-matched control rats (male n = 7; female n = 7). The LV contractile reserve was examined in isolated hearts from each group. We compared LV messenger ribonucleic acid (mRNA) levels of atrial natriuretic factor (ANF), beta-myosin heavy chain, sarcoplasmic reticulum Ca2+-adenosine triphosphatase (ATPase) and Na+-Ca2+ exchanger. Reverse transcriptase polymerase chain reaction was used to identify estrogen receptor transcript in cardiac myocytes and LV tissue., Results: The magnitude of LV hypertrophy (LVH) and systolic wall stress were similar in male and female animals with LVH. Male LVH hearts demonstrated a depressed contractile reserve; in contrast, contractile reserve was preserved in female LVH hearts. The expression of beta-myosin heavy chain and ANF mRNA was greater in male versus female LVH hearts. Sarcoplasmic reticulum Ca2+-ATPase mRNA levels were depressed in male LVH but not in female LVH compared with control rats, and Na+-Ca2+ exchanger mRNA levels were increased similarly in both male and female LVH hearts. Estrogen receptor transcript was detected in both adult male and female cardiac myocytes and LV tissue., Conclusions: There are significant gender differences in the LV adaptation to pressure overload despite a similar degree of LVH and systolic wall stress in male and female rats. There is the potential for estrogen signaling through the adult myocyte estrogen receptor in both male and female rats to contribute to gender differences in gene expression in pathologic hypertrophy.

Cardiac hypertrophy is the fundamental adaptation of the adult heart to mechanical load. Recent work has shown that inhibition of calcineurin activity with cyclosporine suppresses the development of hypertrophy in calcineurin transgenic mice and in in vitro systems of neonatal rat cardiocytes stimulated with peptide growth factors. To test the hypothesis that the calcineurin signaling pathway is critical for load-induced hypertrophy in vivo, we examined the effects of cyclosporine treatment on left ventricular hypertrophy induced by experimental ascending aortic stenosis for 4 weeks in mice. Left ventricular systolic pressure was elevated to a similar level in aortic stenosis mice that were treated with cyclosporine versus no drug. Left ventricular mass and myocyte size were similar in treated and untreated aortic stenosis animals and significantly greater than control animals, showing that cyclosporine treatment does not suppress hypertrophic growth. Both treated and untreated animals showed increased left ventricular expression of the load-sensitive gene atrial natriuretic factor. Calcineurin activity was measured in the left ventricle and the spleen from control mice and aortic stenosis mice treated with cyclosporine versus no drug. Levels of calcineurin activity were similar in the spleens of control and untreated aortic stenosis mice. However, calcineurin activity was severely depressed in left ventricular tissue of untreated aortic stenosis mice compared with control mice and was further reduced by cyclosporine treatment. Thus, pathological hypertrophy and cardiac-restricted gene expression induced by pressure overload in vivo are not suppressed by treatment with cyclosporine and do not appear to depend on the elevation of left ventricular calcineurin activity.

Background: Recombinant human growth hormone (GH) improves in vivo cardiac function in rats with postinfarction heart failure (MI). We examined the effects of growth hormone (14 days of 3.5 mg. kg-1. d-1 begun 4 weeks after MI) on contractile reserve in left ventricular myocytes from rats with chronic postinfarction heart failure., Methods and Results: Cell shortening and [Ca2+]i were measured with the indicator fluo 3 in myocytes from MI, MI+GH, control, and normal animals treated with GH (C+GH) under stimulation at 0.5 Hz at 37 degrees C. Cell length was similar in MI and MI+GH rats (150+/-5 and 157+/-5 microm) and was greater in these groups than in the control and C+GH groups (140+/-4 and 139+/-4 microm, P<0.05). At baseline perfusate calcium of 1.2 mmol/L, myocyte fractional shortening and [Ca2+]i transients were similar among the 4 groups. We then assessed contractile reserve by measuring the increase in myocyte fractional shortening in the presence of high-perfusate calcium of 3.5 mmol/L. In the control and C+GH groups, myocyte fractional shortening and peak systolic [Ca2+]i were similarly increased in the presence of high-perfusate calcium. In the presence of high-perfusate calcium, both myocyte fractional shortening and peak systolic [Ca2+]i were depressed in the MI compared with the control groups. In contrast, myocyte fractional shortening (14.1+/-.9% versus 11.1+/-.9%, P<0.05) and peak systolic [Ca2+]i (647+/-43 versus 509+/-37 nmol/L, P<0.05) were significantly higher in MI+GH than in MI rats and were comparable to controls. Left ventricular myocyte expression of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA-2) and left ventricular SERCA-2 protein levels were increased in MI+GH compared with MI rats., Conclusions: Calcium-dependent contractile reserve is depressed in myocytes from rats with postinfarction heart failure. Long-term growth hormone therapy increases contractile reserve by restoring normal augmentation of systolic [Ca2+]i in myocytes from rats with postinfarction heart failure.

Background: We have previously shown that the acute molecular growth response of new protein synthesis and protein kinase C activation in response to angiotensin II (Ang II) is altered in left ventricular (LV) hypertrophy compared with normal hearts. We have also shown an upregulation of Ang II type 2 (AT2) receptors in hypertrophied hearts relative to controls. Activation of AT2 receptors is proposed to counteract growth effects of AT1 receptor in response to Ang II. Thus, we tested the hypothesis that in hypertrophied hearts, the AT2 receptor mediates inhibitory effects on the new cardiac protein synthesis in response to acute Ang II stimulation., Methods and Results: Flaccid buffer-perfused adult normal and hypertrophied rat hearts were perfused with Ang II 10(-8) mol/L plus prazosin 10(-7) mol/L or Ang II plus the AT2 blocker PD 123319 5x10(-7) mol/L. New protein synthesis was measured by the rate of [3H]phenylalanine incorporation into the LV proteins. In normal hearts, Ang II (n=8) increased the rate of [3H]phenylalanine incorporation by 74+/-27% (P<0.05 versus no drug). Treatment with PD123319 (n=8) did not increase protein synthesis compared with Ang II alone (32+/-11% versus Ang II alone, P=NS). In hypertrophied hearts, Ang II alone (n=6) increased the rate of [3H]phenylalanine incorporation only by 23+/-13% (P=NS versus no drug). In contrast, treatment with PD123319 (n=7) induced a 76+/-21% increase in new LV protein synthesis compared with Ang II alone (P<0.05). AT2 receptor blockade in Ang II-stimulated hypertrophied hearts was associated with enhanced membrane protein kinase C translocation and reduced LV cGMP content., Conclusions: These data support the hypothesis that in adult hypertrophied rat hearts, inhibition of cardiac AT2 receptors, which are upregulated in chronic LV hypertrophy, amplifies the immediate LV growth response to Ang II. This appears to be related to augmented Ang II-stimulated PKC activation and suppression of cGMP signaling.

Background: Atrial natriuretic peptide (ANP) depresses contractility in left ventricular myocytes. Its expression is upregulated in pressure-overloaded hypertrophied hearts; however, the effects of ANP on contractility in hypertrophied myocytes are not known. Our aims were (1) to examine the cellular mechanisms of this depression in contractility in normal myocytes and (2) to test the hypothesis that the effects of ANP on contractility differ in hypertrophied myocytes from rats with ascending aortic stenosis., Methods and Results: We measured the myocyte shortening as an index of contractility, [Ca2+]i with fluo 3, and pHi with seminaphthorhodafluor-1 (SNARF-1). In normal control myocytes (n=26), ANP caused a concentration-dependent depression of contractility and reduction in pHi. In the presence of 10(-6) mol/L ANP, fractional cell shortening was 78+/-5% of baseline (P<0.05) and pHi was reduced by 0.16+/-0.04 U from baseline (P<0.01) without changes in [Ca2+]i. The magnitude of the depression of contraction caused by ANP was similar to that caused by intracellular acidification induced by an NH4Cl pulse. The effects of ANP on contractility and pHi were prevented in the presence of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which inhibits the Na+/H+ exchanger. In hypertrophied myocytes (n=23), ANP did not depress either myocyte contractility or pHi at concentrations of either 10(-8), 10(-7), or 10(-6) mol/L. ANP caused no change in pHi or the [Ca2+]i transient in hypertrophied myocytes. The cGMP level was increased and Na+/H+ exchanger mRNA levels were normal in left ventricles from aortic stenosis rats compared with controls., Conclusions: ANP directly depresses contractility in normal myocytes via intracellular acidification, which decreases myofilament [Ca2+]i sensitivity. In contrast, ANP causes no effects on contractility and pHi in hypertrophied myocytes, suggesting a suppression in the coupling of the ANP-cGMP intracellular signaling pathway to the Na+/H+ exchanger.