Telomerase activity in rat cardiac myocytes is age and gender dependent

Telomerase replaces telomeric repeat DNA lost during the cell cycle, restoring telomere length. This enzyme is present only during cell replication and its activity reflects the extent of proliferation. Whether cardiac myocytes are terminally differentiated cells is still a highly controversial issue, and the possibility of myocyte division is frequently rejected. On this basis, telomerase was measured in pure preparations of myocytes, isolated from rats throughout their lifespan. Fetal and neonatal rat myocytes were used as positive control cells. Contrary to expectation, the authors report that telomerase activity was detectable in pure preparations of young adult, fully mature adult, and senescent ventricular myocytes, defeating the dogma that this cell population is permanent and irreplaceable. Aging decreased 31% telomerase activity in male myocytes. An opposite effect occurred in female myocytes in which this enzyme increased 72%. This differential adaptation between the two genders in the rat model may be relevant to observations in humans; myocyte loss occurs in men as a function of age, whereas myocyte number is preserved in women. The greater growth potential of female myocytes may be critical for the longer lifespan and decreased incidence of heart failure in women.     

Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy

It is generally believed that increase in adult contractile cardiac mass can be accomplished only by hypertrophy of existing myocytes. Documentation of myocardial regeneration in acute stress has challenged this dogma and led to the proposition that myocyte renewal is fundamental to cardiac homeostasis. Here we report that in human aortic stenosis, increased cardiac mass results from a combination of myocyte hypertrophy and hyperplasia. Intense new myocyte formation results from the differentiation of stem-like cells committed to the myocyte lineage. These cells express stem cell markers and telomerase. Their number increased >13-fold in aortic stenosis. The finding of cell clusters with stem cells making the transition to cardiogenic and myocyte precursors, as well as very primitive myocytes that turn into terminally differentiated myocytes, provides a link between cardiac stem cells and myocyte differentiation. Growth and differentiation of these primitive cells was markedly enhanced in hypertrophy, consistent with activation of a restricted number of stem cells that, through symmetrical cell division, generate asynchronously differentiating progeny. These clusters strongly support the existence of cardiac stem cells that amplify and commit to the myocyte lineage in response to increased workload. Their presence is consistent with the notion that myocyte hyperplasia significantly contributes to cardiac hypertrophy and accounts for the subpopulation of cycling myocytes.     

Chronic pressure overload cardiac hypertrophy and failure in guinea pigs: II. Cytoskeletal remodeling

The cytoskeleton is a major regulator of cell shape. To explore potential mechanisms for maladaptation of cardiac myocyte shape in pressure overload-induced congestive heart failure, the abundance and organization of major intra- and extra-myofibrillar cytoskeleton of cardiac myocytes were examined with western blotting and confocal microscopy in guinea pigs with chronic aortic stenosis. It was found that: (1) the amount and distribution of alpha-actinin and myomesin remained unchanged at both the compensated hypertrophy and the congestive heart failure stages; (2) loss of titin was associated with myocyte lengthening in heart failure; (3) desmin protein and filaments in LV myocytes increased progressively with mechanical overload cardiac hypertrophy and subsequent heart failure; (4) a newly developed and validated quantitative confocal microscopic approach disclosed that the microtubule density in isolated LV myocytes increased by 21% at 4 weeks and by 48% 6 months after aortic constriction; (5) at the heart failure stage, microtubule density in LV myocytes showed a statistically significant inverse correlation to the LV maximum +dP/dt and a direct correlation to LV myocyte lengthening; (6) the increased microtubule density in LV myocytes in this model was not due to an increase in total tubulin; and (7) microtubule density in left atrial and right ventricular myocytes also increased when they underwent hypertrophy secondary to left heart failure. These results suggest that the down-regulation of titin and up-regulation of microtubule and desmin filaments may contribute to myocyte lengthening and malfunction in pressure overload congestive heart failure.     

Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure

Chronological myocardial aging is viewed as the inevitable effect of time on the functional reserve of the heart. Cardiac failure in elderly patients is commonly interpreted as an idiopathic or secondary myopathy superimposed on the old heart independently from the aging process. Thus, aged diseased hearts were studied to determine whether cell regeneration was disproportionate to the accumulation of old dying cells, leading to cardiac decompensation. Endomyocardial biopsies from 19 old patients with a dilated myopathy were compared with specimens from 7 individuals of similar age and normal ventricular function. Ten patients with idiopathic dilated cardiomyopathy were also analyzed to detect differences with aged diseased hearts. Senescent cells were identified by the expression of the cell cycle inhibitor p16INK4a and cell death by hairpin 1 and 2. Replication of primitive cells and myocytes was assessed by MCM5 labeling, myocyte mitotic index, and telomerase function. Aged diseased hearts had moderate hypertrophy and dilation, accumulation of p16INK4a positive primitive cells and myocytes, and no structural damage. Cell death markedly increased and occurred only in cells expressing p16INK4a that had significant telomeric shortening. Cell multiplication, mitotic index and telomerase increased but did not compensate for cell death or prevented telomeric shortening. Idiopathic dilated cardiomyopathy had severe hypertrophy and dilation, tissue injury, and minimal level of p16INK4a labeling. In conclusion, telomere erosion, cellular senescence, and death characterize aged diseased hearts and the development of cardiac failure in humans.     

Myocyte growth and cardiac repair

Introduced several decades ago, the dogma persists that ventricular myocytes are terminally differentiated cells and cardiac repair by myocyte regeneration is completely inhibited shortly after birth. On the basis that cardiac myocytes are unable to divide in the adult heart, myocyte growth under physiologic and pathologic conditions is believed to be restricted to cellular hypertrophy. Evidence is presented to indicate that this old paradigm has to be changed to include myocyte replication as a significant component of the cellular processes of ventricular remodeling. Importantly, myocyte death, apoptotic and necrotic in nature, has to be regarded as an additional critical variable of the multifactorial events implicated in the alterations of cardiac anatomy and myocardial structure of the decompensated heart. Methodologies are currently available to recognize and measure quantitatively the contribution of myocyte size, number and death to the adaptation of the overloaded heart and its progression to cardiac failure.     

Increased apoptosis and myocyte enlargement with decreased cardiac mass; distinctive features of the aging male, but not female, monkey heart

We studied gender-specific changes in aging cardiomyopathy in a primate model, Macaca fascicularis, free of the major human diseases, complicating the interpretation of data specific to aging in humans. Left ventricular (LV) weight/body weight decreased, p<0.05, in old males but did not change in old females. However, despite the decrease in LV weight, mean myocyte cross-sectional area in the old males increased by 51%. This increase in myocyte size was not uniform in old males, i.e., it was manifest in only 20-30% of all the myocytes from old males. In old males there was a 4-fold increase in frequency of myocyte apoptosis without any increase in proliferation-capable myocytes assessed by Ki-67 expression. Apoptosis was unchanged in old female monkey hearts, whereas the frequency of myocytes expressing Ki-67 declined 90%. These results, opposite to findings from rodent studies, indicate distinct differences in which male and female monkeys maintain functional heart mass during aging. The old male hearts demonstrated increased apoptosis, which more than offset the myocyte hypertrophy. Interestingly, the hypertrophy was not uniform and there was no significant increase in myocyte proliferation.     

Ventricular loading is coupled with DNA synthesis in adult cardiac myocytes after acute and chronic myocardial infarction in rats.

To determine whether the overload associated with myocardial infarction and ventricular failure in rats is coupled with activation of DNA synthesis in the remaining left and right ventricular myocytes, flow cytometric analysis was performed on myocyte nuclei prepared from both ventricles 7 and 30 days after coronary occlusion. In addition, oral captopril was administered in separate groups of control and experimental rats to establish whether a relation existed between attenuation of ventricular loading and magnitude of DNA synthesis in myocytes. Results demonstrated that left ventricular failure and right ventricular dysfunction at 7 days after infarction were biventricularly associated with marked increases in the number of myocyte nuclei in the G2M phase of the cell cycle. In contrast, the fraction of nuclei in the G0+G1 phase decreased. In comparison with the earlier time point, the 30-day interval was characterized by a significant magnitude of cardiac hypertrophy, a moderate amelioration of ventricular pump function, and a decrease in the percentage of myocyte nuclei in the G2M phase in both ventricles. However, 30 days after infarction, the number of right ventricular myocyte nuclei in the S and G2M phases remained elevated with respect to control animals. Captopril therapy reduced the extent of ventricular loading and the population of myocyte nuclei in the cell cycle at 7 days. In conclusion, DNA synthesis in myocyte nuclei may represent an important adaptive component of the myocardial response to infarction.     

Nuclear targeting of Akt enhances ventricular function and myocyte contractility

Cytoplasmic overexpression of Akt in the heart results in a myopathy characterized by organ and myocyte hypertrophy. Conversely, nuclear-targeted Akt does not lead to cardiac hypertrophy, but the cellular basis of this distinct heart phenotype remains to be determined. Similarly, whether nuclear-targeted Akt affects ventricular performance and mechanics, calcium metabolism, and electrical properties of myocytes is unknown. Moreover, whether the expression and state of phosphorylation of regulatory proteins implicated in calcium cycling and myocyte contractility are altered in nuclear-targeted Akt has not been established. We report that nuclear overexpression of Akt does not modify cardiac size and shape but results in an increased number of cardiomyocytes, which are smaller in volume. Additionally, the heart possesses enhanced systolic and diastolic function, which is paralleled by increased myocyte performance. Myocyte shortening and velocity of shortening and relengthening are increased in transgenic mice and are coupled with a more efficient reuptake of calcium by the sarcoplasmic reticulum (SR). This process increases calcium loading of the SR during relengthening. The enhanced SR function appears to be mediated by an increase in SR Ca2+-ATPase2a activity sustained by a higher degree of phosphorylation of phospholamban. This posttranslational modification was associated with an increase in phospho-protein kinase A and a decrease in protein phosphatase-1. Together, these observations provide a plausible biochemical mechanism for the potentiation of myocyte and ventricular function in Akt transgenic mice. Therefore, nuclear-targeted Akt in myocytes may have important implications for the diseased heart.     

Increased myostatin activity and decreased myocyte proliferation in chronic alcoholic cardiomyopathy

Background: Apoptosis mediates in alcohol‐induced heart damage leading to cardiomyopathy (CMP). Myocyte proliferation may compensate for myocyte loss. Myostatin is upregulated after cardiac damage and by alcohol consumption thereby decreasing myocyte renewal. We assess the potential role of alcohol in inducing myocyte apoptosis as well as in inhibiting myocyte proliferation. Methods: Heart samples were obtained from organ donors, including 22 high alcohol consumers, 22 with hypertension, 8 with other causes of CMP, and 10 healthy donors. Evaluation included medical record with data on daily, recent and lifetime ethanol consumption, chest X‐ray, left ventricular (LV) function assessed by two‐dimensional echocardiography, and LV histology and immunohistochemistry. Apoptosis was evaluated by TUNEL, BAX, and BCL‐2 assays. Myocyte proliferation was evaluated with Ki‐67 assay. Myostatin activity was measured with a specific immunohistochemical assay. CMP was assessed by functional and histological criteria. Results: Alcoholic and hypertensive donors with CMP showed higher apoptotic indices than did their partners without CMP. Myostatin activity was higher in alcoholics than in controls, mainly in those with CMP. The increase in myostatin expression in alcoholic CMP was higher than in other groups. The Ki‐67 proliferation index increased in all groups with CMP compared to those without CMP, with alcoholics showing a lower increase in this proliferation response. Conclusions: Alcohol produces cardiac myocyte loss through apoptosis but also partially inhibits myocyte proliferation through myostatin up‐regulation. The final result may suppose an imbalance in myocyte homeostasis, with a net loss in total ventricular myocyte mass and progressive ventricular dysfunction.     

Comparison of myocardial changes between pressure induced hypertrophy and normal growth in the rat heart

To evaluate differences in tissue composition between hearts with pressure overload hypertrophy and normal hearts of comparable weight, 30 rat hearts with aortic constriction of 4, 10 and 30 days, and nine hearts of sham operated controls were studied. Surgery was performed at age 70 days. Morphometric analysis of myocardial tissue sections revealed (1) myocyte hypertrophy in left ventricular myocardium of hypertrophic hearts was proportional to heart weight, and in normal growth myocyte volume increased in proportion to heart weight; (2) myocyte number in left ventricular myocardium was identical in hypertrophic and normal hearts; (3) non-muscle cell proliferation was proportional to heart weight identically in hypertrophic and normal hearts; (4) volume fractions of myocytes were significantly lower in hypertrophic hearts [0.76(SD 0.05)] than in normal hearts [0.82(0.04)]; (5) volume fractions of all nuclei, myocyte nuclei and non-myocyte nuclei were similar in hypertrophic and normal hearts; (6) measured ventricular DNA content increased with heart weight identically in hypertrophic and normal hearts, and equalled DNA content calculated using the data on tissue composition. Neither right ventricular weight nor right ventricular DNA content were affected by the presence of left ventricular hypertrophy. We conclude that left ventricular hypertrophy due to aortic constriction in the rat resulted in changes of myocardial tissue composition similar to the changes associated with normal growth. Tissue composition of hypertrophic rat hearts corresponds strikingly to that of normal rat hearts with comparable heart weight, although myocardial changes in hypertrophy develop considerably faster than in normal growth.     

Regulation of cardiac remodeling by nitric oxide: focus on cardiac myocyte hypertrophy and apoptosis

Cardiac hypertrophy occurs in pathological conditions associated with chronic increases in hemodynamic load. Although hypertrophy can initially be viewed as a salutary response, ultimately, it often enters a phase of pathological remodeling that may lead to heart failure and premature death. A prevailing concept predicts that changes in gene expression in hypertrophied cardiac myocytes and cardiac myocyte loss by apoptosis contribute to the transition from hypertrophy to failure. In recent years, nitric oxide (NO) has emerged as an important regulator of cardiac remodeling. Specifically, NO has been recognized as a potent antihypertrophic and proapoptotic mediator in cultured cardiac myocytes. Studies in genetically engineered mice have extended these findings to the in vivo situation. It appears that low levels and transient release of NO by endothelial NO synthase exert beneficial effects on the remodeling process by reducing cardiac myocyte hypertrophy, cavity dilation and mortality. By contrast, high levels and sustained production of NO by inducible NO synthase seem to be maladaptive by reducing ventricular contractile function, and increasing cardiac myocyte apoptosis, and mortality. In the future, these novel insights into the role of NO in cardiac remodeling should allow the development of novel therapeutic strategies to treat cardiac remodeling and failure.     

Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression

To determine whether cellular aging leads to a cardiomyopathy and heart failure, markers of cellular senescence, cell death, telomerase activity, telomere integrity, and cell regeneration were measured in myocytes of aging wild-type mice (WT). These parameters were similarly studied in insulin-like growth factor-1 (IGF-1) transgenic mice (TG) because IGF-1 promotes cell growth and survival and may delay cellular aging. Importantly, the consequences of aging on cardiac stem cell (CSC) growth and senescence were evaluated. Gene products implicated in growth arrest and senescence, such as p27Kip1, p53, p16INK4a, and p19ARF, were detected in myocytes of young WT mice, and their expression increased with age. IGF-1 attenuated the levels of these proteins at all ages. Telomerase activity decreased in aging WT myocytes but increased in TG, paralleling the changes in Akt phosphorylation. Reduction in nuclear phospho-Akt and telomerase resulted in telomere shortening and uncapping in WT myocytes. Senescence and death of CSCs increased with age in WT impairing the growth and turnover of cells in the heart. DNA damage and myocyte death exceeded cell formation in old WT, leading to a decreased number of myocytes and heart failure. This did not occur in TG in which CSC-mediated myocyte regeneration compensated for the extent of cell death preventing ventricular dysfunction. IGF-1 enhanced nuclear phospho-Akt and telomerase delaying cellular aging and death. The differential response of TG mice to chronological age may result from preservation of functional CSCs undergoing myocyte commitment. In conclusion, senescence of CSCs and myocytes conditions the development of an aging myopathy.     

Cardiomyopathy of the aging human heart. Myocyte loss and reactive cellular hypertrophy

To determine the effects of aging on the human myocardium, 67 hearts were obtained from individuals who died from causes other than cardiovascular disease. The age interval examined was 17-90 years. Regression analysis demonstrated that the aging process was characterized by a loss of 38 million and 14 million myocyte nuclei/yr in the left and right ventricular myocardium, respectively. This loss in muscle mass was accompanied by a progressive increase in myocyte cell volume per nucleus in both ventricles. Left ventricular myocytes enlarged by 110 microns3/yr, whereas right ventricular myocytes increased by 118 microns3/yr, resulting in a preservation of ventricular wall thickness. However, the cellular hypertrophic response was unable to maintain normal cardiac mass. Left and right ventricular weights decreased by 0.70 and 0.21 g/yr, respectively. In conclusion, loss of cells and enlargement of the remaining myocytes may represent the structural basis for the reduced compensatory capacity of the aged heart and together may contribute to the development of myocardial dysfunction and failure in the elderly.     

Lats2 is a negative regulator of myocyte size in the heart

Mammalian sterile 20-like kinase (Mst)1 plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Because Hippo, a Drosophila homolog of Mst1, forms a signaling complex with Warts, a serine/threonine kinase, which in turn stimulates cell death and inhibits cell proliferation, mammalian homologs of Warts, termed Lats1 and Lats2, may mediate the function of Mst1. We here show that Lats2, but not Lats1, dose-dependently increased apoptosis in cultured cardiac myocytes. Lats2 also dose-dependently reduced [(3)H]phenylalanine incorporation and cardiac myocyte size, whereas dominant negative Lats2 (DN-Lats2) increased them, suggesting that endogenous Lats2 negatively regulates myocyte growth. DN-Lats2 significantly attenuated induction of apoptosis and inhibition of hypertrophy by Mst1, indicating that Lats2 mediates the function of Mst1 in cardiac myocytes. Cardiac specific overexpression of Lats2 in transgenic mice significantly reduced the size of left and right ventricles, whereas that of DN-Lats2 caused hypertrophy in both ventricles. Overexpression of Lats2 reduced left ventricular systolic and diastolic function without affecting baseline levels of myocardial apoptosis. Expression of endogenous Lats2 was significantly upregulated in response to transverse aortic constriction. Overexpression of DN-Lats2 significantly enhanced cardiac hypertrophy and inhibited cardiac myocyte apoptosis induced by transverse aortic constriction. These results suggest that Lats2 is necessary and sufficient for negatively regulating ventricular mass in the heart. Although Lats2 is required for cardiac myocyte apoptosis in response to pressure overload, it was not sufficient to induce apoptosis at baseline. In conclusion, Lats2 affects both growth and death of cardiac myocytes, but it primarily regulates the size of the heart and acts as an endogenous negative regulator of cardiac hypertrophy.     

伊贝沙坦逆转高血压左心室肥厚的细胞学机制

目的探讨伊贝沙坦(IBT)抗高血压左心室肥厚过程中,对心肌细胞凋亡和心肌肌浆网钙泵活性的影响。方法选用16周龄自发性高血压大鼠(SHR)24只,随机分为IBT组(8只)、蒸馏水(DW)组(8只)和SHR0组(8只),另选16只WKY大鼠作为正常对照,随机分为WKY0组(8只)和WKY1组(8只)。IBT组大鼠给予IBT(60 mg.kg-1.d-1)加适量蒸馏水灌胃14周。治疗前后,测量血压和左心室心肌肥厚指数(LVMI),原位末端脱氧核糖核苷酸转移酶介导的dUTP缺口末端标记法检测心肌细胞凋亡,并检测治疗后左心室心肌细胞肌浆网Ca2+-ATP酶活性。结果DW组LVMI、心肌细胞凋亡指数均显著高于WKY组,而IBT组明显低于DW组;DW组Ca2+-ATP酶活性明显低于IBT组及同龄WKY组,IBT组稍低于同龄WKY组;Ca2+-ATP酶活性与LVMI、心肌细胞凋亡指数呈显著负相关,LV-MI与心肌细胞凋亡指数呈显著正相关。结论IBT可能通过调节心肌细胞肌浆网钙泵活性以抑制高血压左心室肥厚过程中心肌细胞凋亡,从而逆转左心室肥厚。     

Angiotensin II and TGF-β in the Development of Cardiac Fibrosis, Myocyte Hypertrophy, and Heart Failure

An explanation of the molecular mechanisms that trigger the development of pathological cardiac fibrosis, myocyte hypertrophy, and heart failure associated with common ailments such as chronic postinfarction has been sought at the bench top and clinic for the past 40years, and is a current topic of intensive investigative activity. During the past several years, awareness of the important role of molecular alterations in the cardiac myocyte and interstitium in cardiac physiology has burgeoned among investigators, and this has led to a focus on the role of cardiac fibrosis and myocyte hypertrophy in the development of heart failure. Among the information garnered from these studies is that growth factors including angiotensin II (AII) and transforming growth factorβ1 (TGF-β1 ), in particular, are believed to be involved in modulation of gene products specifically expressed by cardiac fibroblasts and cardiac myocytes in vitro, and their enhanced presence has been associated with myocardial stress and inappropriate cardiac growth and fibrosis in vivo. Although these growth factors certainly may act on the myocardium alone via specific signaling pathways, we will review evidence that the signals modulated by AII and TGF-β may be coordinated among cardiac fibroblasts and myocytes. In this context, cardiac myocyte hypertrophy and alteration of the cardiac interstitium, i.e., cardiac fibrosis, are examined. This revised version was published online in August 2006 with corrections to the Cover Date.     

The in vivo role of p38 MAP kinases in cardiac remodeling and restrictive cardiomyopathy

Stress-induced mitogen-activated protein kinase (MAP) p38 is activated in various forms of heart failure, yet its effects on the intact heart remain to be established. Targeted activation of p38 MAP kinase in ventricular myocytes was achieved in vivo by using a gene-switch transgenic strategy with activated mutants of upstream kinases MKK3bE and MKK6bE. Transgene expression resulted in significant induction of p38 kinase activity and premature death at 7-9 weeks. Both groups of transgenic hearts exhibited marked interstitial fibrosis and expression of fetal marker genes characteristic of cardiac failure, but no significant hypertrophy at the organ level. Echocardiographic and pressure-volume analyses revealed a similar extent of systolic contractile depression and restrictive diastolic abnormalities related to markedly increased passive chamber stiffness. However, MKK3bE-expressing hearts had increased end-systolic chamber volumes and a thinned ventricular wall, associated with heterogeneous myocyte atrophy, whereas MKK6bE hearts had reduced end-diastolic ventricular cavity size, a modest increase in myocyte size, and no significant myocyte atrophy. These data provide in vivo evidence for a negative inotropic and restrictive diastolic effect from p38 MAP kinase activation in ventricular myocytes and reveal specific roles of p38 pathway in the development of ventricular end-systolic remodeling.