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.     

Adolescent feline heart contains a population of small, proliferative ventricular myocytes with immature physiological properties

Recent studies suggest that rather than being terminally differentiated, the adult heart is a self-renewing organ with the capacity to generate new myocytes from cardiac stem/progenitor cells (CS/PCs). This study examined the hypotheses that new myocytes are generated during adolescent growth, to increase myocyte number, and these newly formed myocytes are initially small, mononucleated, proliferation competent, and have immature properties. Ventricular myocytes (VMs) and cKit(+) (stem cell receptor) CS/PCs were isolated from 11- and 22-week feline hearts. Bromodeoxyuridine incorporation (in vivo) and p16(INK4a) immunostaining were measured to assess myocyte cell cycle activity and senescence, respectively. Telomerase activity, contractions, Ca(2+) transients, and electrophysiology were compared in small mononucleated (SMMs) and large binucleated (LBMs) myocytes. Heart mass increased by 101% during adolescent growth, but left ventricular myocyte volume only increased by 77%. Most VMs were binucleated (87% versus 12% mononucleated) and larger than mononucleated myocytes. A greater percentage of SMMs was bromodeoxyuridine positive (SMMs versus LBMs: 3.1% versus 0.8%; P<0.05), and p16(INK4a) negative and small myocytes had greater telomerase activity than large myocytes. Contractions and Ca(2+) transients were prolonged in SMMs versus LBMs and Ca(2+) release was disorganized in SMMs with reduced transient outward current and T-tubule density. The T-type Ca(2+) current, usually seen in fetal/neonatal VMs, was found exclusively in SMMs and in myocytes derived from CS/PC. Myocyte number increases during adolescent cardiac growth. These new myocytes are initially small and functionally immature, with patterns of ion channel expression normally found in the fetal/neonatal period.     

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.     

Diabetes promotes cardiac stem cell aging and heart failure, which are prevented by deletion of the p66shc gene

Diabetes leads to a decompensated myopathy, but the etiology of the cardiac disease is poorly understood. Oxidative stress is enhanced with diabetes and oxygen toxicity may alter cardiac progenitor cell (CPC) function resulting in defects in CPC growth and myocyte formation, which may favor premature myocardial aging and heart failure. We report that in a model of insulin-dependent diabetes mellitus, the generation of reactive oxygen species (ROS) leads to telomeric shortening, expression of the senescent associated proteins p53 and p16INK4a, and apoptosis of CPCs, impairing the growth reserve of the heart. However, ablation of the p66shc gene prevents these negative adaptations of the CPC compartment, interfering with the acquisition of the heart senescent phenotype and the development of heart failure with diabetes. ROS elicit 3 cellular reactions: low levels activate cell growth, intermediate quantities trigger cell apoptosis, and high amounts initiate cell necrosis. CPC replication predominates in diabetic p66shc-/-, whereas CPC apoptosis and myocyte apoptosis and necrosis prevail in diabetic wild type. Expansion of CPCs and developing myocytes preserves cardiac function in diabetic p66shc-/-, suggesting that intact CPCs can effectively counteract the impact of uncontrolled diabetes on the heart. The recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell disease in which abnormalities in CPCs define the life and death of the heart. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other.     

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.     

Electrophysiologic characteristics of single myocytes isolated from hypertrophied guinea-pig hearts

To determine whether the electrical changes associated with cardiac hypertrophy are due to alterations in the membrane properties of individual hypertrophied cells, we recorded action potentials in single myocytes isolated from normal and hypertrophied hearts. Cardiac hypertrophy was produced by a gradual pressure overload created by placing a band around the ascending aorta in young guinea-pigs (200-250 g). Almost half the animals that developed left ventricular (LHV) hypertrophy also developed evidence of cardiac dysfunction. Action potentials were recorded with standard microelectrodes in single ventricular myocytes isolated by enzymatic dispersion of the heart. The action potential duration at 1 Hz was significantly longer in hypertrophied cells than in control cells. The degree of action potential prolongation in isolated cells did not correlate with the degree of hypertrophy but did correlate with the degree of myocardial disease, the duration being longer in hypertrophied myocytes from dyspneic than in those from non-dyspneic animals. The resting potential was significantly lower in hypertrophied myocytes from dyspneic animals than in hypertrophied cells from non-dyspneic animals or control cells stimulated at 5 Hz. The relationship between the frequency of stimulation (0.33, 1, and 5 Hz) and action potential duration was steeper in hypertrophied than normal myocytes. The mean membrane capacitance (cm) of hypertrophied myocytes increased by 31% over the control value. Thus, isolated hypertrophied myocytes retain the prolonged duration of the action potential and the exaggerated dependence of duration on rate observed in intact hypertrophied muscle. The increased duration of the action potential in hypertrophied cells cannot be readily attributed to the observed increase in cm. Our results indicate that the membrane changes responsible for the altered electrical properties of hypertrophied myocardium are due to an effect of hypertrophy on individual myocytes and that the prolonged duration of the action potential is probably due to changes in active currents flowing during repolarization.     

Increased Contraction of Myocytes Isolated From the Young Spontaneously Hypertensive Rat: Relationship Between Systolic and Diastolic Function

This study was designed to assess heart performance in young (10-week-old) spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, in terms of whole heart function in vivo and mechanics of isolated ventricular myocytes in vitro. The data suggest that left ventricular pressure (LVP) generation is greater, and the maximal velocities of LVP generation and decline are faster in SHR than in WKY. Two-dimensional morphologic measurements show that SHR myocytes are hypertrophied and that augmented contractile function is also present in isolated cells as determined by the extent of shortening and velocity of shortening. Relaxation is also faster at the myocyte level as determined by velocity of relengthening. However, the slope of the relationship between myocyte peak shortening and velocity of relaxation was similar in both groups. These results suggest that hyperdynamic myocyte relengthening may reflect changes in elastic recoil from increased shortening rather than intrinsic changes in cellular mechanisms, which are independent of shortening.     

G1 cyclins are involved in the mechanism of cardiac myocyte hypertrophy induced by angiotensin II

The importance of the cell cycle in proliferating cells is well known, but little is known about the role of cell cycle regulatory proteins in cardiac myocytes, which are fully differentiated cells. The present study determined, in vitro, the effect of angiotensin II (Ang II) treatment of neonatal rat cardiac myocytes on protein levels of cyclins and retinoblastoma gene product (pRb) phosphorylation. The role of G1 cyclin/cdk in Ang II-induced cardiac myocyte hypertrophy by overexpressing cdk inhibitor p21Cip1/Waf1 or p16INK4a was also examined using recombinant adenoviral vectors encoding these genes. Western blot analysis revealed that Ang II stimulated cyclin D1, D2, D3 and A protein levels in cardiac myocytes. Moreover, Ang II phosphorylated pRb on serine 780, which is known to occur in mitotic cells during cell cycle progression. Cultured cardiac myocytes treated with Ang II and infected with either control or recombinant adenovirus indicated that expression of p21 and p16 inhibited Ang II-induced cardiac myocyte hypertrophy, [3H]leucine incorporation into total cellular proteins, and skeletal alpha-actin (SK-A) and atrial natriuretic peptide (ANP) mRNA accumulation. Control virus had no effects on these parameters. These results suggest that G1 cyclins play an important role in cardiac myocyte hypertrophy stimulated by Ang II.     

Overexpression of cdk Inhibitor p16INK4a by adenovirus vector inhibits cardiac hypertrophy in vitro and in vivo: a novel strategy for the gene therapy of cardiac hypertrophy.

Cardiac hypertrophy is one of the serious complications which increase mortality due to cardiovascular diseases. However, only a partial reduction of cardiac hypertrophy has been successful using current drug therapy. We demonstrate here reduction of cardiac hypertrophy in vitro and in vivo using an adenovirus vector encoding cyclin-dependent kinase (cdk) inhibitor p16INK4a. Adenovirus-mediated overexpression of cdk inhibitor p16INK4a completely inhibited cardiac myocyte hypertrophy induced by endothelin (ET)-1, as evaluated by [3H]leucine incorporation into the cells and mRNA levels of skeletal alpha -actin (SK-A) or atrial natriuretic peptide (ANP) as well as by morphometric analyses. We then evaluated whether p16INK4a can suppress left-ventricular (LV) hypertrophy induced by aortic banding (AOB) in rats. Catheter-mediated gene transfer of AxCAp16 was performed according to the method reported by Hajjar et al. LV overload was produced by coarctation of the ascending aorta immediately after inoculation of the heart with adenovirus. Two weeks after the procedure, the left ventricular weight/body weight ratio (LVW/BW) increased in the AOB+LacZ group in comparison to that in controls. However, LVW/BW was identical in the AOB+p16 group and controls. Histologic analysis revealed that p16INK4a inhibited hypertrophy of cardiac myocytes. These results suggest that G1 cell cycle regulators may restrict cardiac hypertrophy, and offer a novel strategy for the gene therapy of cardiac hypertrophy.     

p16基因甲基化在人二倍体成纤维细胞衰老中的作用

目的 研究DNA甲基化与衰老细胞中细胞周期负调控因子p16^MTS1/INK4a高表达的关系。方法 应用甲基化敏感的DNA限制性内切酶与PCR相结合的方法,分析特定位点的甲基化状态。结果 人胚肺二倍体成纤维细胞衰老过程中p16基因表达增高,中年细胞与衰老细胞中p16基因的表达水平分别约为年轻细胞的3倍和10倍。p16基因外显子Ⅰ限制性内切酶SmaⅠ位点的甲基化水平则表现出随增龄而降低的趋势,在年轻细胞、中年细胞中分别约为64％和41％,虽然在衰老细胞中仅为24％,但仍保持一定的水平。结论 p16基因外显子Ⅰ的SmaⅠ位点的甲基化水平改变可能与其在衰老过程中的高表达有一定的关联,其重要性值得进一步研究。     

The phosphoinositide 3-kinase inhibitor LY294002 enhances cardiac myocyte contractility via a direct inhibition of Ik,slow currents

OBJECTIVE: Phosphoinositide 3-kinase (PI3K) is a key component in regulating myocardial growth, survival and contractility. LY294002 and wortmannin are two PI3K inhibitors used widely to establish the role of PI3K. The goal of this study was to examine the effects of acute application of LY294002 and wortmannin on cardiac myocyte contractility and underlying mechanisms. METHODS: Patch-clamp, indo-1 epifluorescence and video-edge detection techniques were used to measure outward K(+) currents, action potentials (AP), Ca(2+) transients and shortening of myocytes isolated from mouse left ventricular free wall. RESULTS: In field-stimulated myocytes, LY294002 (10 micromol/l) increased Ca(2+) transient amplitude by 23%, and cell shortening amplitude by 60% in the absence or presence of wortmannin, while wortmannin alone had no effect. LY294002 (but not wortmannin) prolonged AP duration by specifically inhibiting slowly inactivating K(+) currents (i.e., the 4-aminopyrydine-sensitive I(k,slow1) and the tetraethylammonium-sensitive I(k,slow2)), leading to an increase in sarcoplasmic reticular Ca(2+) levels. It appeared that the AP prolongation was responsible for elevated contractility since AP-clamp of myocytes with prolonged APs (recorded in LY294002-treated myocytes) induced a 29% increase in cell shortening compared with control APs, while LY294002 application did not increase contractility in voltage-clamp studies using either step or AP depolarizations. CONCLUSIONS: The putative PI3K inhibitor LY294002 increases Ca(2+) release and myocyte contractility via direct inhibition of cardiac I(k,slow) and AP prolongation, thus limiting the usefulness of this agent in the analyses of the role of PI3K in heart function.     

Young adult bone marrow-derived endothelial precursor cells restore aging-impaired cardiac angiogenic function

Delivery of young bone marrow-derived stem cells offers a novel approach for restoring the impaired senescent cardiac angiogenic function that may underlie the increased morbidity and mortality associated with ischemic heart disease in older individuals. Recently, we reported that alterations in endothelial cells of the aging heart lead to a dysregulation in the cardiac myocyte platelet-derived growth factor (PDGF)-B-induced paracrine pathway, which contributes to impaired cardiac angiogenic function. Based on these results, we hypothesized that cellular restoration of the PDGF pathway by bone marrow-derived endothelial precursor cells (EPCs) could reverse the aging-associated decline in angiogenic activity. In vitro studies revealed that young murine (3-month-old) bone marrow-derived EPCs recapitulated the cardiac myocyte-induced expression of PDGF-B, whereas EPCs from the bone marrow of aging mice (18-month-old) did not express PDGF-B when cultured in the presence of cardiac myocytes. Transplantation of young, but not old, genetically marked syngeneic bone marrow cells into intact, unirradiated aging mice that populated the endogenous senescent murine bone marrow incorporated into the neovasculature of subsequently transplanted syngeneic neonatal myocardium. Moreover, the young bone marrow-derived EPCs restored the senescent host angiogenic PDGF-B induction pathway and cardiac angiogenesis, with graft survival and myocardial activity in the aging murine host (cardiac allograft viability: 3-month-old controls, 8/8; 18-month-old controls, 1/8; 18-month-old donors receiving bone marrow from 3-month-old mice, 15/16; or 18-month-old mice, 0/6; P<0.05). These results may offer a foundation for the development of novel therapies for the prevention and treatment of cardiovascular disease associated with aging.     

Altered myocardial Ca2+ cycling after left ventricular assist device support in the failing human heart

OBJECTIVES: The objective of the present study was to determine whether improved contractility after left ventricular assist device (LVAD) support reflects altered myocyte calcium cycling and changes in calcium-handling proteins. BACKGROUND: Previous reports demonstrate that LVAD support induces sustained unloading of the heart with regression of pathologic hypertrophy and improvements in contractile performance. METHODS: In the human myocardium of subjects with heart failure (HF), with non-failing hearts (NF), and with LVAD-supported failing hearts (HF-LVAD), intracellular calcium ([Ca(2+)](i)) transients were measured in isolated myocytes at 0.5 Hz, and frequency-dependent force generation was measured in multicellular preparations (trabeculae). Abundance of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA), Na(+)/Ca(2+) exchanger (NCX), and phospholamban was assessed by Western analysis. RESULTS: Compared with NF myocytes, HF myocytes exhibited a slowed terminal decay of the Ca(2+) transient (DT(terminal), 376 +/- 18 ms vs. 270 +/- 21 ms, HF vs. NF, p < 0.0008), and HF-LVAD myocytes exhibited a DT(terminal) that was much shorter than that observed in HF myocytes (278 +/- 10 ms, HF vs. HF-LVAD, p < 0.0001). Trabeculae from HF showed a negative force-frequency relationship, compared with a positive relationship in NF, whereas a neutral relationship was observed in HF-LVAD. Although decreased SERCA abundance in HF was not altered by LVAD support, improvements in [Ca(2+)](i) transients and frequency-dependent contractile function were associated with a significant decrease in NCX abundance and activity from HF to HF-LVAD. CONCLUSIONS: Improvement in rate-dependent contractility in LVAD-supported failing human hearts is associated with a faster decay of the myocyte calcium transient. These improvements reflect decreases in NCX abundance and transport capacity without significant changes in SERCA after LVAD support. Our results suggest that reverse remodeling may involve selective, rather than global, normalization of the pathologic patterns associated with the failing heart.     

Impaired cell shortening and relengthening with increased pacing frequency are intrinsic to the senescent mouse cardiomyocyte

Increased heart rate enhances cardiac contractility and accelerates relaxation. Both the force- and relaxation-frequency relationships are critical to myocardial function, especially during stress, and have been shown to be impaired in senescent myocardium. While senescent myocardium is characterized by decreased sarcoplasmic reticulum calcium ATPase activity, it is unclear if altered calcium regulation is directly responsible for the attenuated contractility and relaxation observed with increasing pacing frequency in aged myocardium. We examined this issue using freshly dissociated left ventricular myocytes, isolated from young adult and senescent mouse hearts. Myocytes were paced from 2 to 9 Hz at 37 degrees C, and cell shortening and [Ca(2+)](i)were simultaneously measured using video edge-detection and fura-2 fluorescence, respectively. In adult myocytes, increasing the pacing rate resulted in a progressive increase in percent cell shortening (CS) (P<0.01). This positive CS-frequency relationship was paralleled by an increase in [Ca(2+)](i)transient amplitude (P<0.05). In contrast, the CS-frequency relationship was blunted in senescent myocytes with no increase in percent CS or [Ca(2+)](i)transient amplitude with increasing pacing rate. With increased pacing, the decreases in time constants (tau) of cell relengthening and Ca(2+)transient decay were much steeper in adult compared to senescent myocytes (P<0.05). This study demonstrates that adult mouse myocytes exhibit augmented intracellular Ca(2+)transient amplitude and enhanced intracellular Ca(2+)removal with increasing pacing frequency, resulting in increased cell shortening and enhanced relengthening with frequency. In contrast, senescent mouse myocytes exhibit impaired calcium handling with increasing pacing frequency, which correlated with impairment of both cell shortening and relengthening.     

Telomerase expression and activity are coupled with myocyte proliferation and preservation of telomeric length in the failing heart

The role and even the existence of myocyte proliferation in the adult heart remain controversial. Documentation of cell cycle regulators, DNA synthesis, and mitotic images has not modified the view that myocardial growth can only occur from hypertrophy of an irreplaceable population of differentiated myocytes. To improve understanding the biology of the heart and obtain supportive evidence of myocyte replication, three indices of cell proliferation were analyzed in dogs affected by a progressive deterioration of cardiac performance and dilated cardiomyopathy. The magnitude of cycling myocytes was evaluated by the expression of Ki67 in nuclei. Ki67 labeling of left ventricular myocytes increased 5-fold, 12-fold, and 17-fold with the onset of moderate and severe ventricular dysfunction and overt failure, respectively. Telomerase activity in vivo is present only in multiplying cells; this enzyme increased 2.4-fold and 3.1-fold in the decompensated heart, preserving telomeric length in myocytes. The contribution of cycling myocytes to telomerase activity was determined by the colocalization of Ki67 and telomerase in myocyte nuclei. More than 50% of Ki67-positive cells expressed telomerase in the overloaded myocardium, suggesting that these myocytes were the morphological counterpart of the biochemical assay of enzyme activity. Moreover, we report that 20--30% of canine myocytes were telomerase competent, and this value was not changed by cardiac failure. In conclusion, the enhanced expression of Ki67 and telomerase activity, in combination with Ki67-telomerase labeling of myocyte nuclei, support the notion that myocyte proliferation contributes to cardiac hypertrophy of the diseased heart.     

Endothelin-1 may act as a negative inotropic agent in cardiac myocytes from young and senescent rats

The contractile response to Endothelin-1 (ET-1) has been investigated in electrically stimulated single cardiac myocytes isolated from hearts of young and senescent rats. ET-1 (0.1-10 nM) exhibited a marked negative inotropic effect in both age groups. ET-1 (1.0 nM) also reduced the occurrence of spontaneous contractile oscillations in young myocytes, suggesting depletion of sarcoplasmic reticulum-Ca(2+) stores. The possibility that ET-1 may have opposite effects on myocardial contractility is discussed.     

bcl-2 overexpression promotes myocyte proliferation

To determine the influence of Bcl-2 on the developmental biology of myocytes, we analyzed the population dynamics of this cell type in the heart of transgenic (TG) mice overexpressing Bcl-2 under the control of the alpha-myosin heavy chain promoter. TG mice and non-TG (wild type, WT) mice were studied at 24 days, 2 months, and 4 months after birth. Bcl-2 overexpression produced a significant increase in the percentage of cycling myocytes and their mitotic index. These effects were strictly connected to the expression of the transgene, as demonstrated in isolated myocytes. The formation of mitotic spindle and contractile ring was identified in replicating cells. These typical aspects of mitosis were complemented with the demonstration of karyokinesis and cytokinesis to provide structural evidence of cell division. Apoptosis was low at all ages and was not affected by Bcl-2. The higher cell replication rate in TG was conditioned by a decrease in the expression of the cell-cycle inhibitors, p21(WAF1) and p16(INK4a), and by an increase in Mdm2-p53 complexes. In comparison with WT, TG had 0.4 x 10(6), 0.74 x 10(6), and 1.2 x 10(6) more myocytes in the left ventricle at 24 days, 2 months, and 4 months, respectively. Binucleated myocytes were 12% and 25% larger in WT than in TG mice at 2 and 4 months of age. Taken together, these observations reveal a previously uncharacterized replication-enhancing function of Bcl-2 in myocytes in vivo in the absence of stressful conditions.     

Reduced swelling-activated Cl(-) current densities in hypertrophied ventricular myocytes of rabbits with heart failure

OBJECTIVE: Hypertrophied myocytes of failing hearts have prolonged action potential durations. It is unknown how the swelling-activated Cl(-) current (I(Cl,swell)) affects the abnormal AP configuration. METHODS: We studied I(Cl,swell) in ventricular myocytes isolated from failing and age-matched normal rabbit hearts. We applied whole-cell patch-clamp methodology and activated I(Cl,swell) by lowering tonicity of the superfusate. RESULTS: Neither with ruptured-patch nor with amphotericin B perforated-patch, whole-cell clamp we found I(Cl,swell) active under isotonic conditions in either the normal or the hypertrophied failing heart (HFH) myocytes. I(Cl,swell) caused AP shortening and resting membrane potential (V(m)) depolarization in an osmotic gradient-dependent fashion. However, in the HFH myocytes swelling-induced AP changes were significantly smaller, even though the cells underwent the same relative change in planar cell surface area. Voltage-clamp experiments revealed that in HFH myocytes I(Cl,swell) current density was approximately 50% reduced. CONCLUSION: Reduced I(Cl,swell) densities in HFH myocytes cause limited AP shortening and V(m) depolarization upon swelling of the cells.     

Comparison of lipoprotein lipase activity in heart myocytes and perfused hearts

This study was initiated to compare lipoprotein lipase activity in isolated heart myocytes and the heparin residual compartment of perfused hearts from adult rats. Heart lipoprotein lipase activity was divided into two fractions by 1 min of heparin perfusion. Heparin-residual and myocyte lipoprotein lipase activity were lower in hearts obtained from fasted compared to fed rats. In each case, the myocyte enzyme activity was 55 to 60% of heparin-residual levels. The difference between myocyte and heparin-residual activity may be a consequence of the time and treatment required to isolate cells in that long-term in vitro exposure of heart tissue to heparin also reduces residual activity. In vivo treatment with endotoxin decreased both heparin-residual and myocyte lipoprotein lipase activities; whereas, colchicine administration increased both activities compared to saline injected rats. In this latter experiment heart heparin-residual and myocyte lipoprotein lipase activities were positively correlated (r = 0.90). The results indicate that in the mature heart intracellular lipoprotein lipase activity is primarily associated with myocytes.     

Stem cell niches in the adult mouse heart

Cardiac stem cells (CSCs) have been identified in the adult heart, but the microenvironment that protects the slow-cycling, undifferentiated, and self-renewing CSCs remains to be determined. We report that the myocardium possesses interstitial structures with the architectural organization of stem cell niches that harbor long-term BrdU-retaining cells. The recognition of long-term label-retaining cells provides functional evidence of resident CSCs in the myocardium, indicating that the heart is an organ regulated by a stem cell compartment. Cardiac niches contain CSCs and lineage-committed cells, which are connected to supporting cells represented by myocytes and fibroblasts. Connexins and cadherins form gap and adherens junctions at the interface of CSCs-lineage-committed cells and supporting cells. The undifferentiated state of CSCs is coupled with the expression of alpha(4)-integrin, which colocalizes with the alpha(2)-chain of laminin and fibronectin. CSCs divide symmetrically and asymmetrically, but asymmetric division predominates, and the replicating CSC gives rise to one daughter CSC and one daughter committed cell. By this mechanism of growth kinetics, the pool of primitive CSCs is preserved, and a myocyte progeny is generated together with endothelial and smooth muscle cells. Thus, CSCs regulate myocyte turnover that is heterogeneous across the heart, faster at the apex and atria, and slower at the base-midregion of the ventricle.