Federica del Monte

Akt Activation Preserves Cardiac Function and Prevents Injury After Transient Cardiac Ischemia In Vivo

Background—The serine-threonine kinase Akt is activated by several ligand-receptor systems previously shown to be cardioprotective. Akt activation reduces cardiomyocyte apoptosis in models of transient ischemia. Its role in cardiac dysfunction or infarction, however, remains unclear. Methods and Results—We examined the effects of a constitutively active Akt mutant (myr-Akt) in a rat model of cardiac ischemia-reperfusion injury. In vivo gene transfer of myr-Akt reduced infarct size by 64% and the number of apoptotic cells by 84% (P <0.005 for each). Ischemia-reperfusion injury decreased regional cardiac wall thickening as well as the maximal rate of left ventricular pressure rise and fall (+dP/dt and −dP/dt). Akt activation restored regional wall thickening and +dP/dt and −dP/dt to levels seen in sham-operated rats. To better understand this benefit, we examined the effects of myr-Akt on hypoxic cardiomyocyte dysfunction in vitro. myr-Akt prevented hypoxia-induced abnormalities in cardiomyocyte calcium transients and shortening. Akt activation also enhanced sarcolemmal expression of Glut-4 in vivo and increased glucose uptake in vitro to the level seen with insulin treatment. Conclusions—Akt activation exerts a powerful cardioprotective effect after transient ischemia that probably reflects its ability to both inhibit cardiomyocyte death and improve function of surviving cardiomyocytes. Akt may represent an important nodal target for therapy in ischemic and other heart disease.

Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair

Adult mammalian hearts respond to injury with scar formation and not with cardiomyocyte proliferation, the cellular basis of regeneration. Although cardiogenic progenitor cells may maintain myocardial turnover, they do not give rise to a robust regenerative response. Here we show that extracellular periostin induced reentry of differentiated mammalian cardiomyocytes into the cell cycle. Periostin stimulated mononucleated cardiomyocytes to go through the full mitotic cell cycle. Periostin activated αV, β1, β3 and β5 integrins located in the cardiomyocyte cell membrane. Activation of phosphatidylinositol-3-OH kinase was required for periostin-induced reentry of cardiomyocytes into the cell cycle and was sufficient for cell-cycle reentry in the absence of periostin. After myocardial infarction, periostin-induced cardiomyocyte cell-cycle reentry and mitosis were associated with improved ventricular remodeling and myocardial function, reduced fibrosis and infarct size, and increased angiogenesis. Thus, periostin and the pathway that it regulates may provide a target for innovative strategies to treat heart failure.

Restoration of Contractile Function in Isolated Cardiomyocytes From Failing Human Hearts by Gene Transfer of SERCA2a

BACKGROUND Failing human myocardium is characterized by abnormal relaxation, a deficient sarcoplasmic reticulum (SR) Ca(2+) uptake, and a negative frequency response, which have all been related to a deficiency in the SR Ca(2+) ATPase (SERCA2a) pump. METHODS AND RESULTS To test the hypothesis that an increase in SERCA2a could improve contractile function in cardiomyocytes, we overexpressed SERCA2a in human ventricular myocytes from 10 patients with end-stage heart failure and examined intracellular Ca(2+) handling and contractile function. Overexpression of SERCA2a resulted in an increase in both protein expression and pump activity and induced a faster contraction velocity (26.7+/-6.7% versus 16.6+/-2.7% shortening per second, P<0.005) and enhanced relaxation velocity (32. 0+/-10.1% versus 15.1+/-2.4%, P<0.005). Diastolic Ca(2+) was decreased in failing cardiomyocytes overexpressing SERCA2a (270+/-26 versus 347+/-30 nmol/L, P<0.005), whereas systolic Ca(2+) was increased (601+/-38 versus 508+/-25 nmol/L, P<0.05). In addition, the frequency response was normalized in cardiomyocytes overexpressing SERCA2a. CONCLUSIONS These results support the premise that gene-based therapies and targeting of specific pathways in human heart failure may offer a new modality for the treatment of this disease.

Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure.

Background —Left ventricular failure is commonly preceded by a period of hypertrophy. Intriguingly, many of the signaling pathways that have been implicated in the regulation of hypertrophy, including the 3 mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, stress-activated protein kinase, and p38), protein phosphatase, calcineurin, and the protein kinase Akt and its target glycogen synthase kinase-3 (GSK-3), also regulate the apoptotic response. Methods and Results —To understand the mechanisms that might regulate the progression of heart failure, we analyzed the activity of these signaling pathways in the hearts of patients with advanced heart failure, patients with compensated cardiac hypertrophy, and normal subjects. In patients with hypertrophy, neither the MAPK nor the Akt/GSK-3 pathways were activated, and the dominant signaling pathway was calcineurin. In failing hearts, calcineurin activity was increased but less so than in the hypertrophied hearts, and all 3 MAPKs and Akt were activated (and, accordingly, GSK-3&bgr; was inhibited), irrespective of whether the underlying diagnosis was ischemic or idiopathic cardiomyopathy. Conclusions —In the failing heart, there is a clear prohypertrophic activity profile, likely occurring in response to increased systolic wall stress and neurohormonal mediators. However, with the activation of these hypertrophic pathways, potent proapoptotic and antiapoptotic signals may also be generated. Therapies directed at altering the balance of activity of these signaling pathways could potentially alter the progression of heart failure.

Titin Isoform Switch in Ischemic Human Heart Disease

Background—Ischemia-induced cardiomyopathy usually is accompanied by elevated left ventricular end-diastolic pressure, which follows from increased myocardial stiffness resulting from upregulated collagen expression. In addition to collagen, a main determinant of stiffness is titin, whose role in ischemia-induced left ventricular stiffening was studied here. Human heart sarcomeres coexpress 2 principal titin isoforms, a more compliant N2BA isoform and a stiffer N2B isoform. In comparison, normal rat hearts express almost no N2BA titin. Methods and Results—Gel electrophoresis and immunoblotting were used to determine the N2BA-to-N2B titin isoform ratio in nonischemic human hearts and nonnecrotic left ventricle of coronary artery disease (CAD) patients. The average N2BA-to-N2B ratio was 47:53 in severely diseased CAD transplanted hearts and 32:68 in nonischemic transplants. In normal donor hearts and donor hearts with CAD background, relative N2BA titin content was ≈30%. The titin isoform shift in CAD transplant hearts coincided with a high degree of modifications of cardiac troponin I, probably indicating increased preload. Immunofluorescence microscopy on CAD transplant specimens showed a regular cross-striated arrangement of titin and increased expression of collagen and desmin. Force measurements on isolated myofibrils revealed reduced passive-tension levels in sarcomeres of CAD hearts with high left ventricular end-diastolic pressure compared with sarcomeres of normal hearts. In a rat model of ischemia-induced myocardial infarction (left anterior descending coronary artery ligature), 43% of animals, but only 14% of sham-operated animals, showed a distinct N2BA titin band on gels. Conclusions—A titin isoform switch was observed in chronically ischemic human hearts showing extensive remodeling, which necessitated cardiac transplantation. The shift, also confirmed in rat hearts, caused reduced titin-derived myofibrillar stiffness. Titin modifications in long-term ischemic myocardium could impair the ability of the heart to use the Frank-Starling mechanism.

Cardiomyogenesis in the Adult Human Heart

Rationale: The ability of the human heart to regenerate large quantities of myocytes remains controversial, and the extent of myocyte renewal claimed by different laboratories varies from none to nearly 20% per year. Objective: To address this issue, we examined the percentage of myocytes, endothelial cells, and fibroblasts labeled by iododeoxyuridine in postmortem samples obtained from cancer patients who received the thymidine analog for therapeutic purposes. Additionally, the potential contribution of DNA repair, polyploidy, and cell fusion to the measurement of myocyte regeneration was determined. Methods and Results: The fraction of myocytes labeled by iododeoxyuridine ranged from 2.5% to 46%, and similar values were found in fibroblasts and endothelial cells. An average 22%, 20%, and 13% new myocytes, fibroblasts, and endothelial cells were generated per year, suggesting that the lifespan of these cells was approximately 4.5, 5, and 8 years, respectively. The newly formed cardiac cells showed a fully differentiated adult phenotype and did not express the senescence-associated protein p16INK4a. Moreover, measurements by confocal microscopy and flow cytometry documented that the human heart is composed predominantly of myocytes with 2n diploid DNA content and that tetraploid and octaploid nuclei constitute only a small fraction of the parenchymal cell pool. Importantly, DNA repair, ploidy formation, and cell fusion were not implicated in the assessment of myocyte regeneration. Conclusions: Our findings indicate that the human heart has a significant growth reserve and replaces its myocyte and nonmyocyte compartment several times during the course of life.

Myocyte Turnover in the Aging Human Heart

Rationale: The turnover of cardiomyocytes in the aging female and male heart is currently unknown, emphasizing the need to define human myocardial biology. Objective: The effects of age and gender on the magnitude of myocyte regeneration and the origin of newly formed cardiomyocytes were determined. Methods and Results: The interaction of myocyte replacement, cellular senescence, growth inhibition, and apoptosis was measured in normal female (n=32) and male (n=42) human hearts collected from patients 19 to 104 years of age who died from causes other than cardiovascular diseases. A progressive loss of telomeric DNA in human cardiac stem cells (hCSCs) occurs with aging and the newly formed cardiomyocytes inherit short telomeres and rapidly reach the senescent phenotype. Our data provide novel information on the superior ability of the female heart to sustain the multiple variables associated with the development of the senescent myopathy. At all ages, the female heart is equipped with a larger pool of functionally competent hCSCs and younger myocytes than the male myocardium. The replicative potential is higher and telomeres are longer in female hCSCs than in male hCSCs. In the female heart, myocyte turnover occurs at a rate of 10%, 14%, and 40% per year at 20, 60, and 100 years of age, respectively. Corresponding values in the male heart are 7%, 12%, and 32% per year, documenting that cardiomyogenesis involves a large and progressively increasing number of parenchymal cells with aging. From 20 to 100 years of age, the myocyte compartment is replaced 15 times in women and 11 times in men. Conclusions: The human heart is a highly dynamic organ regulated by a pool of resident hCSCs that modulate cardiac homeostasis and condition organ aging.

Cardiac angiogenic imbalance leads to peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is an often fatal disease that affects pregnant women who are near delivery, and it occurs more frequently in women with pre-eclampsia and/or multiple gestation. The aetiology of PPCM, and why it is associated with pre-eclampsia, remain unknown. Here we show that PPCM is associated with a systemic angiogenic imbalance, accentuated by pre-eclampsia. Mice that lack cardiac PGC-1α, a powerful regulator of angiogenesis, develop profound PPCM. Importantly, the PPCM is entirely rescued by pro-angiogenic therapies. In humans, the placenta in late gestation secretes VEGF inhibitors like soluble FLT1 (sFLT1), and this is accentuated by multiple gestation and pre-eclampsia. This anti-angiogenic environment is accompanied by subclinical cardiac dysfunction, the extent of which correlates with circulating levels of sFLT1. Exogenous sFLT1 alone caused diastolic dysfunction in wild-type mice, and profound systolic dysfunction in mice lacking cardiac PGC-1α. Finally, plasma samples from women with PPCM contained abnormally high levels of sFLT1. These data indicate that PPCM is mainly a vascular disease, caused by excess anti-angiogenic signalling in the peripartum period. The data also explain how late pregnancy poses a threat to cardiac homeostasis, and why pre-eclampsia and multiple gestation are important risk factors for the development of PPCM.

Type 1 Phosphatase, a Negative Regulator of Cardiac Function

ABSTRACT Increases in type 1 phosphatase (PP1) activity have been observed in end stage human heart failure, but the role of this enzyme in cardiac function is unknown. To elucidate the functional significance of increased PP1 activity, we generated models with (i) overexpression of the catalytic subunit of PP1 in murine hearts and (ii) ablation of the PP1-specific inhibitor. Overexpression of PP1 (threefold) was associated with depressed cardiac function, dilated cardiomyopathy, and premature mortality, consistent with heart failure. Ablation of the inhibitor was associated with moderate increases in PP1 activity (23%) and impaired β-adrenergic contractile responses. Extension of these findings to human heart failure indicated that the increased PP1 activity may be partially due to dephosphorylation or inactivation of its inhibitor. Indeed, expression of a constitutively active inhibitor was associated with rescue of β-adrenergic responsiveness in failing human myocytes. Thus, PP1 is an important regulator of cardiac function, and inhibition of its activity may represent a novel therapeutic target in heart failure.

Restoration of Diastolic Function in Senescent Rat Hearts Through Adenoviral Gene Transfer of Sarcoplasmic Reticulum Ca2+-ATPase

BACKGROUND Senescent hearts are characterized by diastolic dysfunction and a decrease in sarcoplasmic reticulum (SR) Ca(2+)-ATPase protein (SERCA2a). METHODS AND RESULTS To test the hypothesis that an increase in SERCA2a could improve cardiac function in senescent rats (age 26 months), we used a catheter-based technique of adenoviral gene transfer to achieve global myocardial transduction of SERCA2a in vivo. Adult rat hearts aged 6 months and senescent rat hearts infected with an adenovirus containing the reporter gene beta-galactosidase were used as controls. Two days after infection, parameters of systolic and diastolic function were measured in open-chest rats. Cardiac SERCA2a protein and ATPase activity were significantly decreased in senescent hearts compared with adult rats (Delta -30+/-4% and -49+/-5%) and were restored to adult levels after infection with Ad.SERCA2a. At baseline, left ventricular systolic pressure and +dP/dt were unaltered in senescent hearts; however, diastolic parameters were adversely affected with an increase in the left ventricular time constant of isovolumic relaxation and diastolic pressure (Delta +29+/-9% and +38+/-12%) and a decrease in -dP/dt (Delta -26+/-11%). Overexpression of SERCA2a did not significantly affect left ventricular systolic pressure but did increase +dP/dt (Delta +28+/-10%) in the senescent heart. Overexpression of SERCA2a restored the left ventricular time constant of isovolumic relaxation and -dP/dt to adult levels. Infection of senescent hearts with Ad.SERCA2a markedly improved rate-dependent contractility and diastolic function in senescent hearts. CONCLUSIONS These results support the hypothesis that decreased Ca(2+)-ATPase activity contributes to the functional abnormalities observed in senescent hearts and demonstrates that Ca(2+) cycling proteins can be targeted in the senescent heart to improve cardiac function.