Our findings indicate that myocyte apoptosis develops during the transition from hypertrophy to early failure in mice with chronic biomechanical stress and support the hypothesis that the disruption of normal myocyte anchorage to adjacent extracellular matrix and cells, a process called anoikis, may signal apoptosis.

Abstract-Mouse myocyte contractility and the changes induced by pressure overload are not fully understood. We studied contractile reserve in isolated left ventricular myocytes from mice with ascending aortic stenosis (AS) during compensatory hypertrophy (4-week AS) and the later stage of early failure (7-week AS) and from control mice. ] o , 25°C), the amplitude of myocyte shortening and peak-systolic [Ca 2ϩ ] i in 7-week AS were not different from those of controls, whereas contraction, relaxation, and the decline of [Ca 2ϩ ] i transients were slower. In response to the challenge of high [Ca 2ϩ ] o , fractional cell shortening was severely depressed with reduced peak-systolic [Ca 2ϩ ] i in 7-week AS compared with controls. In response to rapid pacing stimulation, cell shortening and peak-systolic [Ca 2ϩ ] i increased in controls, but this response was depressed in 7-week AS. In contrast, the responses to both challenge with high [Ca 2ϩ ] o and rapid pacing in 4-week AS were similar to those of controls. Although protein levels of Na ϩ -Ca 2ϩ exchanger were increased in both 4-week and 7-week AS, the ratio of SR Ca 2ϩ -ATPase to phospholamban protein levels was depressed in 7-week AS compared with controls but not in 4-week AS. This was associated with an impaired capacity to increase sarcoplasmic reticulum Ca 2ϩ load during high work states in 7-week AS myocytes. In hypertrophied failing mouse myocytes, depressed contractile reserve is related to an impaired augmentation of systolic [Ca 2ϩ ] i and SR Ca 2ϩ load and simulates findings in human failing myocytes. (Circ Res. 2000;87:588-595.) Key Words: myocytes Ⅲ contractility Ⅲ sarcoplasmic reticulum Ⅲ Ca 2ϩ -ATPase Ⅲ heart failure Ⅲ hypertrophy T he regulation of contractility is under intense investigation with the use of transgenic mice. 1,2 The mouse cardiovascular system differs from that of humans and larger mammals, including the rapid heart rate, faster myofibrillar ATPase activity and sarcoplasmic reticulum (SR) Ca 2ϩ uptake, 2,3 and higher mechanical performance per unit ventricular mass. 4 To interpret findings in genetically manipulated mice, it is critical to understand the properties of normal mouse myocyte contractility and the changes induced by clinically relevant stimuli such as chronic load. We reported that mice with ascending aortic stenosis (AS) develop compensated hypertrophy (4-week AS) and the later stage of early heart failure (7-week AS). 4,5 The aim of the present study was to examine contractile reserve in myocytes from AS mice in transition from hypertrophy to early heart failure. We measured myocyte contraction and [Ca 2ϩ ] i transients in left ventricular (LV) myocytes from normal, 4-week AS, and 7-week AS mice in response to the challenge of stepped increases in [Ca 2ϩ ] o . In separate experiments, we examined the response to rapid pacing stimulation. Although only subtle abnormalities in the time course of contraction and the [Ca 2ϩ ] i transients are present at baseline, contractile reserve is depressed in ...

The angiotensin II type 2 (AT2) receptor is upregulated in the left ventricle in heart failure, but its pathophysiological roles in vivo are not understood. In the present study, AT2 receptors were expressed in transgenic (TG) mice using the ventricular-specific myosin light-chain (MLC-2v) promoter. In TG compared with nontransgenic (NTG) mice, in vivo left ventricular (LV) systolic pressure and peak +dP/dt were depressed while LV diastolic pressure was elevated (P < 0.05). Echocardiography showed severely depressed LV fractional shortening, increased systolic and diastolic dimensions, and wall thinning (P < 0.05). Confocal and electron microscopy studies revealed an increase in the size of myocytes and interstitial spaces as well as an increase in interstitial collagen, disruption of the Z-band, and changes in cytochrome c localization. The changes were most prominent in the highest-expressing TG line, which implies a dose-response relationship. AT2 overexpression was also directly associated with the increase of phosphorylated protein levels of PKC-alpha, PKC-beta, and p70S6 kinase. These data demonstrate that ventricular myocyte-specific expression of AT2 receptors promotes the development of dilated cardiomyopathy and heart failure in vivo.

Neuregulins and their erbB receptors are essential for cardiac development and postulated to be cardioprotective in the presence of injury in the postnatal heart. We tested the hypothesis that the development of doxorubicin-induced cardiotoxicity in vivo is more severe in mice with heterozygous knockout of the neuregulin-1 gene (NRG-1(+/-)) compared with wild-type mice (WT). Three-month old NRG-1(+/-) and WT mice were injected with a single dose of doxorubicin (20 mg/kg ip). Survival was analyzed by the Kaplan-Meier approach. Left ventricular (LV) function and signaling pathways were analyzed 4 days after treatment. Fifteen days after treatment, survival was significantly lower in doxorubicin-treated NRG-1(+/-) mice (NRG-1(+/-)-Dox) compared with doxorubicin-treated WT mice (WT-Dox) (15% vs. 33%, P < 0.01). LV mass was significantly lower in NRG-1(+/-)-Dox but not in WT-Dox compared with nontreated animals. LV systolic pressure and LV midwall fractional shortening were significantly lower in NRG-1(+/-)-Dox compared with WT-Dox mice. LV protein levels of NRG-1, erbB2, and erbB4 receptors were similar in WT-Dox and NRG-1(+/-)-Dox mice. However, levels of phosphorylated erbB2, Akt, and ERK-1/2 were significantly decreased in NRG-1(+/-)-Dox compared with WT-Dox mice. A significant decrease in phosphorylated P70S6K levels was also observed in NRG-1(+/-)-Dox compared with nontreated NRG-1(+/-) mice. These results demonstrate that heterozygous knockout of the neuregulin-1 gene worsens survival and LV function in the presence of doxorubicin-induced cardiac injury in vivo. This is associated with the depression of activation of the erbB2 receptor as well as Akt, p70S6K, and ERK-1/2 pathways.

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(NRG1) is a potential therapeutic agent for the treatment of doxorubicin (Dox)-induced heart failure. NRG1, however, activates the erbB2 receptor, which is frequently overexpressed in breast cancers. It is, therefore, important to understand how NRG1, via erbB2, protects the heart against Dox cardiotoxicity. Here, we studied NRG1-erbB2 signaling in Dox-treated mice hearts and in isolated neonatal rat ventricular myocytes (NRVM). Male C57BL/6 mice were treated with recombinant NRG1 before and daily after a single dose of Dox. Cardiac function was determined by catheterization. Two-week survival was analyzed by the Kaplan-Meier method. Cardiac troponins [cardiac troponin I (cTnI) and cardiac troponin T (cTnT)] and phosphorylated Akt protein levels were determined in mice hearts and in NRVM by Western blot analysis. Activation of caspases and ubiquitinylation of troponins were determined in NRVM by caspase assay and immunoprecipitation. NRG1 significantly improved survival and cardiac function in Dox-treated mice. NRG1 reduced the decrease in cTnI, cTnT, and cardiac troponin C (cTnC) and maintained Akt phosphorylation in Dox-treated mice hearts. NRG1 reduced the decrease in cTnI and cTnT mRNA and proteins in Dox-treated NRVM. Inhibition of erbB2, phosphoinositide 3-kinase (PI3K), Akt, and mTOR blocked the protective effects of NRG1 on cTnI and cTnT in NRVM. NRG1 significantly reduced Dox-induced caspase activation, which degraded troponins, in NRVM. NRG1 reduced Doxinduced proteasome degradation of cTnI. NRG1 attenuates Dox-induced decrease in cardiac troponins by increasing transcription and translation and by inhibiting caspase activation and proteasome degradation of troponin proteins. NRG1 maintains cardiac troponins by the erbB2-PI3K pathway, which may lessen Dox-induced cardiac dysfunction.ErbB2; troponin proteins; signaling NEUREGULIN-1 (NRG1)-ErbB2 signaling is essential for cardiac development and maintaining adult cardiac function (26, 29). ErbB2 was initially detected as an oncogenic variant that was overexpressed in several tumor types (15). Trastuzumab, a humanized monoclonal antibody that binds to and blocks erbB2, significantly reduces recurrence and early mortality in patients with breast cancer who overexpress erbB2 (27,34). A significant increase in congestive heart failure was reported, however, when the anti-erbB2 antibody trastuzumab was used in combination with the chemotherapy drug doxorubicin (Dox) (34). Thus inhibition of erbB2 signaling in patients who receive concurrent therapy with Dox causes an increased risk of cardiotoxicity. We hypothesized, accordingly, that activation of erbB2 signaling by NRG1 may mitigate cardiac dysfunction.Multiple isoforms of NRG1 are synthesized in the endocardium and the endothelium of cardiac vasculature (7,20,24). NRG1 activates its receptors erbB2 and erbB4 on cardiomyocytes (11). NRG1 promotes hypertrophy and proliferation of cardiomyocytes through activation of erbB2 and erbB4 and protects cardiomyocytes from apoptosis (20,24,38). The soluble recombinan...

Abstract-To examine the contribution of sarcoplasmic reticulum Ca 2ϩ 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 2ϩ ] i transients were higher in TG than in WT 7-week AS mice under baseline conditions (0. uptake. 3,4 Enhanced contractility has also been reported in otherwise normal TG mice not subjected to pathological stimuli. 5,6 However, many short-term interventions that improve cardiac contractility, such as adrenergic stimulation, are associated with adverse long-term effects on survival and progression of heart failure. 7-10 Therefore, it is controversial whether chronic upregulation of SERCA2a in vivo will favorably modify the development of early heart failure in animals with biomechanical load.To delineate the contribution of SERCA2a to contractile dysfunction during transition from compensatory hypertrophy to early failure, we subjected TG mice overexpressing the SERCA2a transgene 5 to chronic left ventricular (LV) pressure overload caused by ascending aortic stenosis (AS). We recently reported that mice with AS develop compensated hypertrophy (4-week AS) and the later stage of early heart failure (7-week AS). [11][12][13] In vivo LV systolic pressure generation is increased and contractility is preserved in 4-week AS mice, but they are depressed in 7-week AS mice concomitant with a reduction in SERCA2a protein levels. 11,12 In LV myocytes, contractile reserve and the capacity to increase sarcoplasmic reticulum (SR) Ca 2ϩ load are depressed in 7-week AS mice, but not in 4-week AS mice. 13 In the present study, we show that TG expression of SERCA2a in AS mice, which prevents the fall in SERCA2a levels observed by 7-week AS in wild-type (WT) mice, maintains contractile function and the capacity to increase SR Ca 2ϩ load at high work states. The magnitude of hypertrophy itself is not modified by TG SERCA2a expression. These data suggest that a defective SR Ca 2ϩ loading plays a critical role in the onset of early heart failure in animals with biomechanical overload. Materials and Methods Animal PreparationTG mice overexpressing rat SERCA2a transgene were produced as described previously. 5 AS surgery was performed in the ascending Original

Previous epidemiologic data demonstrate that cardiovascular (CV) morbidity and mortality may occur decades after ionizing radiation exposure. With increased use of proton and carbon ion radiotherapy and concerns about space radiation exposures to astronauts on future long-duration exploration-type missions, the long-term effects and risks of low-dose charged particle irradiation on the CV system must be better appreciated. Here we report on the long-term effects of whole-body proton (1H; 0.5 Gy, 1 GeV) and iron ion (56Fe; 0.15 Gy, 1GeV/nucleon) irradiation with and without an acute myocardial ischemia (AMI) event in mice. We show that cardiac function of proton-irradiated mice initially improves at 1 month but declines by 10 months post-irradiation. In AMI-induced mice, prior proton irradiation improved cardiac function restoration and enhanced cardiac remodeling. This was associated with increased pro-survival gene expression in cardiac tissues. In contrast, cardiac function was significantly declined in 56Fe ion-irradiated mice at 1 and 3 months but recovered at 10 months. In addition, 56Fe ion-irradiation led to poorer cardiac function and more adverse remodeling in AMI-induced mice, and was associated with decreased angiogenesis and pro-survival factors in cardiac tissues at any time point examined up to 10 months. This is the first study reporting CV effects following low dose proton and iron ion irradiation during normal aging and post-AMI. Understanding the biological effects of charged particle radiation qualities on the CV system is necessary both for the mitigation of space exploration CV risks and for understanding of long-term CV effects following charged particle radiotherapy.