Cardiac-Specific IGF-1 Expression Attenuates Dilated Cardiomyopathy in Tropomodulin-Overexpressing Transgenic Mice
To test the hypothesis that early interventional treatment with insulin-like growth factor-1 (IGF-1) alleviates subsequent development of dilated cardiomyopathy, cardiac-specific IGF-1 expression was introduced by selective cross-breeding into a transgenic mouse model of heart failure that displays phenotypic characteristics of severe dilation. Hemodynamic, structural, and cellular parameters of the heart were compared between nontransgenic, tropomodulin-overexpressing cardiomyopathic, and the hybrid tropomodulin/IGF-1-overexpressing mice. Beneficial effects of IGF-1 were apparent by multiple indices of cardiac structure and function, including normalization of heart mass, anatomy, hemodynamics, and apoptosis. IGF-1 expression also acted as a proliferative stimulus as evidenced by calculated increases in myocyte number as well as expression of Ki67, a nuclear marker of cellular replication. Cellular analyses revealed that IGF-1 inhibited characteristic cardiomyocyte elongation in dilated hearts and restored calcium dynamics comparable to that observed in normal cells. Collectively, these results provide novel information regarding the ability of IGF-1 to inhibit progression of cardiomyopathic disease in a defined model system and suggest that heart failure may benefit from early interventional IGF-1 treatment.
Dilated cardiomyopathy in humans is characterized by scattered myocardial damage with focal areas of replacement fibrosis. Myocyte apoptosis, necrosis, and regeneration have also been implicated in this cardiac disease.1– 4⇓⇓⇓ However, cell death exceeds cell multiplication, resulting in the onset of terminal failure.3,4⇓ Additionally, myocyte lengthening and myofibrillar disarray1,5⇓ contribute to the depression in ventricular performance. A mouse model that mimics the structural and functional characteristics of human dilated cardiomyopathy has been established by overexpressing tropomodulin (Tmod), an actin filament regulatory protein6 specifically in the myocardium.7–10⇓⇓⇓ Tmod-overexpressing transgenic (TOT) mice exhibit alterations in Ca2+ handling with significant increases in resting levels of Ca2+ that precede the expansion in cavitary volume, suggesting that impaired Ca2+ regulation may play a primary role in the development of the cardiomyopathy.9,10⇓ Elevation in Ca2+ concentration has been implicated in apoptotic and necrotic cell death,11,12⇓ raising the possibility that similar cellular mechanisms may be operative in the TOT mouse. Conversely, Ca2+ activates calmodulin-dependent kinases-I and -IV as well as calcineurin, promoting myocyte hypertrophy.13–15⇓⇓ Activation of calcineurin has been implicated in TOT pathogenesis,9,16⇓ but the role of apoptosis in the development of the TOT cardiomyopathy was not examined.
In an attempt to counteract the deterioration of cardiac structure and function leading to dilated cardiomyopathy, the TOT line was crossbred with homozygous transgenic mice overexpressing insulin-like growth factor-1 (IGF-1) in cardiac myocytes17 to create Tmod-IGF-1-overexpressing (TIGFO) mice. This genetic approach was followed in order to interfere in a specific and direct manner with the development of heart failure in a mouse model. Exogenous administration of IGF-1 would affect the entire organism, complicating interpretation of the results. Because both Tmod and IGF-1 transgenes are regulated by the α -myosin heavy chain promoter, effects of cardiac-specific IGF-1 expression could be determined. The rationale for this strategy was based on the ability of IGF-1 to favor the alignment and organization of myofibrils in the cytoplasm18 and to interfere with myocyte apoptosis and necrosis triggered by the formation of oxidative stress.19 Reactive oxygen represents the distal event in Ca2+-mediated cell death.19–21⇓⇓ Low levels of oxidative damage are coupled with apoptosis and high levels with necrosis.22,23⇓ IGF-1 attenuates the generation of reactive oxygen species by limiting angiotensin II formation24 and, thereby, cytosolic Ca2+.20 Importantly, IGF-1 overexpression increases myocyte regeneration and the total number of cells in the heart. This adaptation occurs postnatally and is enhanced with age.17 Therefore, we investigated several factors involved in the ability of IGF-1 to rescue the TOT mouse from the development of a dilated myopathy and premature death.
Materials and Methods
Transgenic Mouse Breeding and Selection
Creation and breeding descriptions have been previously published for both TOT-7 (Sussman lab, Cincinnati, Ohio) and IGF-17 (Anversa lab, Valhalla, NY) overexpressing lines. Transgenic mice were housed in a temperature-controlled, germ-free barrier facility that was AAALAC-approved. All experiments were approved by the Children’s Hospital Research Foundation Animal Care Review Board. A detailed description of the breeding and maintenance of the lines is obtainable in an expanded Materials and Methods section that can be found in the online data supplement available at http://www.circresaha.org.
Ventricular Function and Anatomy
Mice were anesthetized with tribromoethanol (1.2%, 0.2 mL, IP). The carotid artery was cannulated with a microtip pressure transducer (SPR-671, Millar) connected to a chart recorder. The catheter was advanced into the left ventricle (LV) for the evaluation of LV pressures and + and −dP/dt in the closed chest preparation.19,25,26⇓⇓ After collection of hemodynamic measurements, the abdominal aorta was cannulated, the heart was arrested in diastole with CdCl2, and the myocardium was perfused with 10% phosphate-buffered formalin. The left ventricular chamber was fixed at a pressure equal to the in vivo measured end-diastolic pressure. The heart was excised and cardiac weights were recorded. Longitudinal intracavitary axis of LV was measured and 3 transversely cut slices, from the base, midregion, and apex, were obtained. The midsection was utilized to evaluate LV thickness and chamber diameter. Longitudinal axis and chamber diameter were used to compute cavitary volume according to the Dodge equation.25,26⇓ Wall thickness in diastole, chamber radius, and end-diastolic pressure were employed to calculate diastolic stress.19,25,26⇓⇓ Systolic wall stress was computed from echocardiographic measurements of wall thickness and chamber radius in systole and determination of LV systolic pressure at euthanasia. Echocardiography was performed using a Agilent Sonos 5500 equipped with a 15-Mhz linear transducer.7
Detailed descriptions of the protocols for terminal deoxynucleotidyl transferase assay (TUNEL) and in situ ligation are provided in the online data supplement.
A detailed description of the protocol used for preparation of samples and analysis of Ki67 labeling is provided in the online data supplement.
Myocyte Isolation for Morphometry
Details of myocyte isolation for morphometry are provided in the online data supplement.
Myocyte nuclei were stained by propidium iodide and the fraction of mononucleated, binucleated, and multinucleated cells was determined by examining 500 cells from each left ventricle. Myocyte length and width were measured with a computerized image analysis system (Media Cybernetics): 200 binucleated and 50 mononucleated and multinucleated myocytes from each left ventricle were assessed. Because isolated cells assume a cross-sectional area that resembles a flattened ellipse, the ratio of the minor to the major axis of the ellipse was obtained by confocal microscopy. Cell volume was calculated assuming an elliptical cross section with the major axis equivalent to cell width while the minor axis was computed from the measured ratios.17,27⇓
Cross sections of LV were stained with hematoxylin and eosin and examined at ×1000 with a reticule containing 42 sampling points to determine the volume fraction of myocytes and interstitium.17,27⇓ The total volume of myocytes in LV was calculated from the product of LV volume and the volume percent of myocytes. The volume fraction of myocytes and the proportion of mononucleated, binucleated, and multinucleated myocytes were utilized to compute the volume percent of each myocyte class in the myocardium.17,27⇓ The number of mononucleated, binucleated, and multinucleated cells in the LV was calculated from the quotient of their aggregate volume and corresponding myocyte cell volume. The mural number of myocytes was determined from their numerical density per mm2 of myocardium and ventricular wall thickness.1
Samples were prepared for immunoblot analysis as previously described.9 Antibodies were obtained to detect total Akt (Cell Signaling Technologies), phosphospecific (phospho)-Akt473 (Biosource), sarcoplasmic reticulum calcium ATPase (SERCA), and sodium calcium (Na+-Ca2+) exchanger (both from Affinity Bioreagents Inc). Chemifluorescent signals were quantitated by densitometric analysis with Imagequant software of scans obtained using a Storm 860 (Molecular Dynamics).
A detailed description of the protocol used for confocal line scan calcium imaging is provided in the online data supplement.
Details for statistical analysis of results are provided in the online data supplement.
Ventricular Anatomy and Hemodynamics Are Normalized in TIGFOs
Increased heart weight, LV weight, and RV weight in TOTs were essentially normalized by expression of IGF-1 (Table 1). Gross parameters of cardiac anatomy were not statistically different between NTG and TIGFO. Although RV weight-to-body weight ratio was corrected by IGF-1, heart and LV weight-to-body weight ratios were not completely reduced to control values in TIGFO hearts (28%, P<0.03; 24%, P<0.04; data not shown). Conversely, free wall and septal thickness in TIGFOs were similar to control mice, as were the longitudinal axis, chamber diameter, and cavitary volume. Finally, the 16% (P<0.01) decrease in mural number of myocytes in TOTs was completely reversed by IGF-1.
TIGFO mice show a significant improvement of cardiac function compared with TOT mice (Table 1). LV end-diastolic pressure decreased 21% (P<0.001) whereas LV systolic pressure, LV +dP/dt, and LV −dP/dt increased 39% (P<0.001), 112% (P<0.001), and 92% (P<0.001), respectively. Similarly, LV diastolic pressure increased 71% (P<0.001) in TIGFO mice. Echocardiographic analyses (not shown) exhibited good correlation with the anatomic and hemodynamic measurements presented in Table 1.
Increased Myocyte Apoptosis and Necrosis in TOT Hearts Is Eliminated in TIGFOs
Left ventricular myocytes were studied to determine whether IGF-1 expression interfered with activation of cell death by apoptosis or necrosis (Figure 1). Myocyte apoptosis by TUNEL assay (P<0.001) was nearly 4-fold higher in TOT (638±102 per 106 cells) than NTG mice (155±50 per 106 cells), although cell necrosis did not differ between the 2 groups of animals (165±40 versus 148±38 per 106 cells, respectively). Similarly, myocyte necrosis did not vary between NTG and TIGFO (141±41 per 106 cells). Importantly, apoptotic cell death was comparable in NTG and TIGFO (171±46 per 106 cells), indicating that IGF-1 prevented the increase in apoptosis detected in TOTs. It is relevant to emphasize that essentially identical values for myocyte apoptosis were measured by 2 independent methods, the TUNEL assay and hairpin 1 probe (Figure 1), confirming the validity of the TUNEL technique.
Akt activation level was assessed in ventricular heart samples from 6-week-old mice by quantitative immunoblotting using antibody to phospho-Akt473 (Figure 2). Akt activation was increased in all 3 transgenic samples (TOT, IFG, and TIGFO) relative to the NTG control. Phosoho-Akt473 level in the TOT sample was increased 2.9±0.2-fold, presumably due to stress-response-related activation of Akt at this time point. In comparison, the IGF and TIGFO samples showed phospho-Akt473 levels of 3.4±0.2 and 4.3±0.4, respectively, compared with NTG. Differences in Akt activity between the NTG and 3 transgenic groups were all significant (P<0.02).
Ki67 Labeling Is Elevated in TIGFOs
Expression of Ki67 protein in left ventricular myocyte nuclei was measured to evaluate whether myocyte proliferation was implicated in the favorable cardiac restructuring of TIGFO mice. (Figure 3). Ki67 is present only in nuclei of cycling cells as a marker of the late G1-M phase.28 There are no examples of Ki67-positive cells that do not replicate.29 In comparison with NTG, IGF-1 increased the number of cycling myocytes in IGF-expressing transgenics by 2.1-fold (1690±570 versus 3575±155, respectively) and by 3.5-fold in TIGFO mice (5910±1460 per 106 cells). The fraction of myocytes labeled by Ki67 was low in TOT mice (1210±310 per 106 cells).
Myocyte Size and Number Are Normalized in TIGFOs
Measurements of cell death and Ki67 labeling of myocytes provided an accurate evaluation of what was occurring to this cell population at the time of euthanasia; however, single time point analyses are not representative of a phenomenon developing for 6 to 8 weeks since birth. To establish whether ongoing cell death detected by the TUNEL assay and hairpin 1 as well as myocyte replication identified by Ki67 were indicative of a long-term processes (cell dropout and proliferation, respectively), the total number of myocytes in the left ventricle was measured morphometrically. Acquisition of this information required measurement of average volume for myocytes, which was obtained by 3-dimensional optical section reconstruction (Figure 4). The proportion of mononucleated, binucleated, and multinucleated myocytes was essentially identical in all groups studied. Distribution of nuclei within cardiomyocytes was 6.1±2.0% mononucleated (n=28), 91.5±2.2% binucleated (n=28), and 2.4±1.2% multinucleated (n=28). Overexpression of Tmod alone resulted in a 10% (NS), 50% (P<0.001), and 34% (P<0.001) increase in the mean volume of mononucleated, binucleated, and multinucleated myocytes (Table 2). In contrast, sizes of mononucleated, binucleated and multinucleated myocytes were comparable in NTG and TIGFO mice. Hypertrophy of TOT myocytes was due to increased cell length, consistent with the severe degree of ventricular dilation, but IGF-1 prevented cell elongation by preserving the cell length-to-cell diameter ratio in TIGFOs. Calculation of total myocyte number in the left ventricle reveals a 16% (P<0.001) reduction in myocytes relative to NTGs for TOTs aged 6 to 8 weeks. Conversely, myocyte number increased 15% (P<0.001) in TIGFO mouse. When TOT and TIGFO mice were compared, the latter had a left ventricle with 37% (P<0.001) more myocytes.
Calcium Dynamics of Isolated Cardiomyocytes Are Normalized in TIGFOs
Measurement of intracellular calcium concentration resulting from field stimulation of individual cells was performed by line scan confocal microscopy (Figure 5). Calcium dynamics were compared between NTG, TOT, and TIGFO cardiomyocytes paced at frequencies of 0.5, 1.0, and 2.0 Hz. As frequency stimulation rate rises to 2.0 Hz, a significant elevation of diastolic calcium level and decreased amplitude of calcium release is evident in TOT cardiomyocytes compared with NTG cells (see online Table 1 in the online data supplement available at http://www.circresaha.org). In contrast, the response of TIGFO cells with respect to calcium levels was comparable to NTG. Quantitation of results for cells paced at 1 Hz (Figure 5) demonstrates significant prolongation of time required for peak calcium release in TOT cardiomyocytes (137.5±70.7 ms) compared with NTG cells (58.6±22.9 ms). Similarly, time required to achieve 50% calcium reuptake (t50) is also significantly prolonged in TOT cardiomyocytes (647.8±90.7) relative to NTG cells (345.1±131.1). Both time-to-peak calcium and t50 values are normalized in TIGFO cells (55.4± 21.4 and 334.6±119.3, respectively) comparable to values obtained with NTG cardiomyocytes.
The molecular basis for restoration of calcium dynamics was examined by quantitative immunoblot analysis for both SERCA and Na+-Ca2+ exchanger that participate in calcium reuptake and extrusion, respectively (Figure 6). SERCA protein level in TOT mice was decreased 1.5-fold compared with normal NTG samples, whereas Na+-Ca2+ exchanger levels were increased 1.9-fold. These changes were statistically significant for both SERCA (P= 0.047) and Na+-Ca2+ exchanger (P= 0.015). TIGFO mice showed normalization of protein expression with SERCA decreased only 1.1-fold and Na+-Ca2+ exchanger increased 1.35-fold relative to NTG, neither of which were statistically significant changes (P=0.44 and 0.33, respectively). Thus, restoration of calcium dynamics in the TIGFO mice correlates with normalization of major calcium handling protein levels.
Several etiologic factors have been identified in the onset and development of dilated cardiomyopathy in TOT mice. These include abnormalities in Ca2+ handling, activation of calcineurin, changes in Tmod:actin filament stoichiometry, impaired mechanical coupling due to alterations in the intercalated disks, and thin-filament disarray.6,8–10,16,30⇓⇓⇓⇓⇓ Ventricular dilation and mural thinning occurs only by myocyte lengthening, cell loss, wall restructuring with side-to-side slippage of myocytes, or by their combination.31 As shown here, each of these parameters contributes to TOT cardiac pathogenesis. Moreover, myocyte hypertrophy was the exclusive form of growth in response to Tmod overexpression, but was characterized by a marked decrease in cell diameter-to-cell length ratio. On the basis of these observations, TOTs are affected by decompensated eccentric hypertrophy at the ventricular and cellular level.31 The genetic introduction of IGF-1 in TIGFOs interfered with all the structural components of ventricular remodeling seen in TOT hearts. Myocyte hypertrophy, modification in cell shape, myocyte slippage, and cell death were prevented by IGF-1. The ability of IGF-1 to inhibit cell death, apoptotic and necrotic in nature, has previously been shown in experimental ischemic cardiomyopathy in vivo25,26⇓ and in vitro studies of adult myocytes exposed to mechanical deformations mimicking the condition of ventricular dilation.32 Mitigation of cardiomyopathic disease in TIGFOs could not be attributed to decreased Tmod protein expression, because Tmod expression level was comparable between TOT and TIGFO hearts by quantitative immunoblot analysis (data not shown). Beneficial effects of IGF-1 on TOT heart structure were also accompanied by significant improvement in cardiac performance.
IGF-1 expression opposed development of myocyte hypertrophy and cell elongation that, in combination with the preservation of cell viability, almost completely abolished expansion of cavitary volume in TOTs. Additionally, elevation in left ventricular end diastolic pressure and diastolic wall stress, which are the major determinants of myocyte lengthening,33 were essentially corrected by IGF-1. Similarly, depressed systolic function and the decrease in myocyte cross-sectional area were normalized in TIGFOs. Reduction in the number of myocytes within the ventricular wall resulting in TOT myopathy from cell slippage and/or cell death did not occur in TIGFOs.
Myocyte death by apoptosis was increased several-fold in TOTs. The methodology utilized here to recognize and measure this type of cell death allowed us to establish that Ca2+-dependent DNase I was implicated in DNA fragmentation.11,12⇓ This observation strongly suggests that a link existed between abnormalities in Ca2+ handling and the initiation of apoptosis in TOTs, consistent with results showing increased diastolic calcium and decreased calcium amplitude in TOT cardiomyocytes compared with normal or TIGFO cells (online Table 1 and Figure 5). IGF-1 activates phosphatidylinositol 3-kinase (PI3-K), which in turn, phosphorylates the downstream effector molecule Akt34–36⇓⇓; the Akt/PI3-K pathway is involved in the survival of various cell types.34 Moreover, constitutive overexpression of IGF-1 improves myocyte performance, enhancing shortening velocity and cellular compliance.37 In combination, these effects of IGF-1 on myocyte survival and mechanical behavior may explain the beneficial impact of IGF-1 on anatomy and hemodynamics in TIGFO hearts. In this regard, the exogenous administration of IGF-1 ameliorates cardiac functions in animal models of coronary artery disease38–41⇓⇓⇓ and in patients affected by idiopathic dilated cardiomyopathy with modest degree of ventricular decompensation.42 Although apoptosis, Ca2+ dynamics, and cell size were similar in TIGFO and WT mice, ventricular dilation and cardiac function were not completely normalized in the double transgenic mice. This may reflect the in series arrangement of the larger number of cells in the left ventricle of these animals and residual alterations in the mechanical behavior of myocytes, respectively.
IGF-1 enhances growth and viability of myocytes.17,19,25,26⇓⇓⇓ Downregulation of IGF-1 postnatally is characterized by a rapid decline in myocyte replication, whereas upregulation of IGF-1 is associated with activation of the cell cycle and cell division under conditions of stress.43,44⇓ The mitogenic effect of IGF-1 on myocytes seems to depend on its capacity to negatively regulate p53 through the induction of mdm2,24,32⇓ and downregulate p53-dependent cell cycle inhibitors such as p21Cip1/Waf1.45 Additionally, IGF-1 increases telomerase activity, enhancing cell growth.46,47⇓ The higher number of myocytes in the left ventricle of TIGFOs strongly suggests, but does not prove, that myocyte proliferation occurs in these transgenic animals because decreased apoptosis in TIGFOs could also account for the larger number of myocytes. To search for unequivocal evidence of myocyte division, the number of cycling myocytes was identified by Ki67 labeling. Ki67 antigen, a nuclear and nucleolar protein tightly associated with somatic cell division, is detectable cells in G1, S, G2, prophase, and metaphase but decreases rapidly in anaphase and telophase and is absent in G0.28 Although the function of Ki67 remains to be identified in detail, it has been suggested that this nuclear protein interferes with p53 activation and inhibition of cell cycle progression.29 Currently, Ki67 is the most reliable and specific marker of cell proliferation. In contrast to BrdU and thymidine labeling, which recognize only the S-phase and are implicated in DNA repair,28 Ki67 detects essentially all the phases of the cell cycle and is not involved in DNA repair. Thus, Ki-67 results indicate myocyte proliferation in TIGFO mice.
In conclusion, forced expression of IGF-1 rescued the TOT mouse, normalizing the anatomical and functional properties of the myopathic heart. The increased myocyte number in TIGFO hearts associated with Ki67 labeling of myocytes raises the possibility that cell replication played a significant role in the correction of the abnormality in wall thickness, cavitary volume, and ventricular hemodynamics. Whether cell multiplication, inhibition of apoptosis, or both act as the prevailing mechanism of myocardial repair in the TOT mouse remains to be determined. Decreased cell death, prevention of cell lengthening, and side-to-side slippage of myocytes within the wall by IGF-1 are all relevant factors for preservation of cardiac size, shape, and function to prevent dilated cardiomyopathy.
This work was supported by NIH grants HL-38132, HL-39902, HL-43023, AG-15756, HL-66923, AG-17042 (to Piero Anversa), HL-65577 (to Annarosa Leri), HL-65573 (to Jan Kajstura), HL58224, HL66035, and HL67245 (to Mark Sussman). Mark Sussman is a recipient of an Established Investigator Award from the American Heart Association.
Original received December 12, 2001; revision received February 13, 2002; accepted February 13, 2002.
- ↵Beltrami CA, Finato N, Rocco M, Feruglio GA, Puricelli C, Cigola E, Sonnenblick EH, Olivetti G, Anversa P. The cellular basis of dilated cardiomyopathy in humans. J Mol Cell Cardiol. 1991; 27: 291–305.
- ↵Kajstura J, Leri A, Finato N, Di Loreto C, Beltrami CA, Anversa P. Myocyte proliferation in end-stage cardiac failure in humans. Proc Natl Acad Sci U S A. 1998; 95: 8801–8805.
- ↵Guerra S, Leri A, Wang X, Finato N, Di Loreto C, Beltrami CA, Kajstura J, Anversa P. Myocyte death in the failing human heart is gender dependent. Circ Res. 1999; 85: 856–866.
- ↵Schaper J, Froede TA, Hein ST, Buck A, Hashizume H, Speiser B, Friedl A, Bleese N. Impairment of the myocardial ultrastructure and changes in the cytoskeleton in dilated cardiomyopathy. Circulation. 1991; 83: 504–514.
- ↵Weber AM, Pennise CR, Babcock GG, Fowler VM. Tropomodulin caps the pointed ends of actin filaments. J Cell Biol. 1994; 127: 1627–1635.
- ↵Sussman MA, Baqué S, Uhm CS, Daniels MP, Price RL, Simpson D, Terracio L, Kedes L. Altered expression of tropomodulin in cardiomyocytes disrupts the sarcomeric structure of myofibrils. Circ Res. 1998; 82: 94–105.
- ↵Sussman MA, Welch S, Walker A, Klevitsky R, Hewett TE, Witt SA, Kimball TR, Price R, Lim HW, Molkentin JD. Hypertrophic defect unmasked by calcineurin expression in asymptomatic tropomodulin overexpressing transgenic mice. Cardiovasc Res. 2000; 46: 90–101.
- ↵Didenko VV, and Hornsby PJ. Presence of double-strand breaks with single-base 3‘ overhangs in cells undergoing apoptosis but not necrosis. J Cell Biol. 1996; 135: 1369–1376.
- ↵Passier R, Zeng H, Frey N, Naya FJ, Nicol RL, McKinsey TA, Overbeek P, Richardson JA, Grant ST, Olson EN. CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo. J Clin Invest. 1999; 105: 1395–1406.
- ↵De Windt LJ, Lim HW, Bueno OF, Liang Q, Delling U, Braz JC, Glascock BJ, Kimball TF, Del Monte F, Haajar RJ, Molkentin JD. Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A. 2001; 98: 3322–3327.
- ↵Rothermel BA, McKinsey TA, Vega RB, Nicol RL, Mammen P, Yang J, Antos CL, Shelton JM, Bassel-Duby R, Olson EN, Williams RS. Myocyte-enriched calcineurin-interacting protein, MCIP 1, inhibits cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A. 2000; 98: 3328–3333.
- ↵Sussman MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, Colbert MC, Gualberto A, Wieczorek DF, Molkentin JD. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science. 1998; 281: 1690–1693.
- ↵Reiss K, Cheng W, Ferber A, Kajstura J, Li P, Li B, Olivetti G, Homcy CJ, Baserga R, Anversa P. Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice. Proc Natl Acad Sci U S A. 1996; 93: 8630–8635.
- ↵Donath MY, Zapf J, Eppenberger-Eberhardt M, Froesch EF, Eppenberger HM. Insulin-like growth factor 1 stimulates myofibril development and decreases smooth muscle actin of adult cardiomyocytes. Proc Natl Acad Sci U S A. 1994; 91: 1686–1690.
- ↵Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A, Anversa P. IGF-1 overexpression inhibits development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress. Diabetes. 2001; 50: 1414–1424.
- ↵Leri A, Liu Y, Wang X, Kajstura J, Malhotra A, Meggs LG, Anversa P. Overexpression of insulin-like growth factor-1 attenuates the myocyte renin-angiotensin in transgenic mice. Circ Res. 1999; 84: 752–762.
- ↵Li B, Setoguchi M, Wang X, Andreoli AM, Malhotra A, Kajstura J, Anversa P. Insulin-like growth factor-1 attenuates the detrimental impact of non-occlusive coronary artery constriction on the heart. Circ Res. 1999; 84: 1007–1019.
- ↵Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci. U S A. 2001; 98: 10344–10349.
- ↵Ehler E, Horowits R, Zuppinger C, Price RL, Rerriard E, Leu M, Caroni P, Sussman M, Eppenberger HM, Perriard JC. Alterations at the intercalated disk associated with the absence of muscle LIM protein. J Cell Biol. 2001; 153: 763–772.
- ↵Anversa P, Olivetti G, Meggs LG, Sonnenblick EH, Capasso JM. Cardiac anatomy and ventricular loading after myocardial infarction. Circulation. 1997; 87: VII-22–VII-27.
- ↵Kulik G, Weber MJ. Akt-dependent, and independent survival signaling pathways utilized by insulin-like growth factor 1. Mol Cell Biol. 1998; 18: 6711–6718.
- ↵Yamashita K, Kajstura J, Discher DJ, Wasserlauf BJ, Bishopric NH, Anversa P, Webster KA. Reperfusion-activated Akt kinase prevents apoptosis in transgenic mouse hearts overexpressing insulin-like growth factor-1. Circ Res. 2001; 88: 609–614.
- ↵Camper-Kirby D, Welch S, Walker A, Shiraishi I, Setchell KDR, Schaefer E, Kajstura J, Anversa P, Sussman MA. Myocardial Akt activation and gender: increased nuclear activity in females versus males. Circ Res. 2001; 88: 1020–1027.
- ↵Redaelli G, Malhotra A, Li B, Li P, Sonnenblick EH, Hofmann PA, Anversa P. Effects of constitutive overexpression of insulin-like growth factor-1 on the mechanical characteristics and molecular properties of ventricular myocytes. Circ Res. 1998; 82: 594–603.
- ↵Buerke M, Murohara T, Skurk C, Tomaselli K, Lefer AM. Cardioprotective effect of insulin-like growth factor 1 in myocardial ischemia followed by reperfusion. Proc Natl Acad Sci U S A. 1995; 92: 8031–8035.
- ↵Cittadini A, Stromer H, Katz SE, Clark R, Moses AC, Morgan JP, Douglas PS. Differential cardiac effects of growth hormone and insulin-like growth factor-1 in the rat: a combined in vivo and in vitro evaluation. Circulation. 1996; 93: 800–809.
- ↵Stromer H, Cittadini A, Douglas PS, Morgan JP. Exogenously administered growth hormone and insulin-like growth factor-1 alter intracellular Ca2+ handling and enhance cardiac performance: in vitro evaluation in the isolated isovolumic buffer-perfused rat heart. Circ Res. 1996; 79: 227–236.
- ↵Duerr RL, McKirnan MD, Gim RD, Clark RG, Chien KR, Ross J. Cardiovascular effects of insulin-like growth factor-1 and growth hormone in chronic left ventricular failure in rat. Circulation. 1996; 93: 2188–2196.
- ↵Kang SS, Kwon T, Kwon DY, Do SI. Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit. J Biol Chem. 1999; 274: 13085–13090.