Variability of the cardiomyocyte ploidy in normal human hearts

We have performed cytophotometry for DNA in isolated myocytes of the left ventricle from 16 men, aged 19–39 years, who died from various non-cardiac or pulmonary causes. The mean ploidy of myocytes varied from 3.2–3.9 c to 6.6–7.3 c in different layers of the anterior wall of the left ventricle (where c is the haploid DNA content measured by cytophotometry in Feulgenstained preparations). There was no correlation between the layers. The percentage of binuclear cells varied from 25 to 86% and correlated in every layer with the mean ploidy value of the whole myocyte population. Approximate calculation of total ploidy revealed low values in the ventricles of some individuals, and high values in others. Averaging the values for all the hearts studied obscures this variation. Mean myocyte ploidy in different layers of the anterior wall was similar: in the external layer it was 5.1±0.3 c, in the middle layer 5.5±0.3 c and in the inner layer 4.8±0.4 c. The mean percentage of binuclear myocytes in these three layers was also similar, being 61±3%, 63±4% and 54±5%, respectively. Myocyte ploidy in tissue from the posterior wall of the left ventricle also varied, but was always higher than for the same layer of the anterior wall in the same ventricle. We propose that high or low myocyte ploidy, as well as different proportions of mono- and binucleate cells, can be a factor affecting the course and result of cardiac pathology in the absence of any changes of myocyte genome determined during early ontogenesis and representing a stable characteristic of the individual.     

Cumulative indices of DNA synthesizing myocytes in different compartments of the working myocardium and conductive system of the rat’s heart muscle following extensive left ventricle infarction

Ten successive³H-thymidine injections at 12h intervals (which is a little shorter than the adult heart myocyte S phase) were performed for labeling of the majority of cardiac myocytes synthesizing DNA at any moment of such a 5 days experiment. In the hearts of control unoperated rats ten-fold repeated³H-thymidine administration results in labeling of 2–3% myocyte nuclei, in both atria, ca. 1% of the specialized muscle cell nuclei in the atrioventricular conductive system, only occasional muscle cells being labeled in the working ventricular myocardium. When ten successive³H-thymidine injections were made between the 5th and 10th days following extended left ventricle infarction, the percentage of labeled myocytes in left and right atria reaches, respectively, 51.4±4.4% and 34.7±3.6%. In the left ventricle labeled muscle nuclei are accumulated predominantly (9.3±2.1%) within the thin subepicardial layer of the surviving myofibers, while myofibers located in other perinecrotic areas contained only 1.3±0.5% labeled muscle nuclei. The number of these nuclei in the atrioventricular system remains at the level observed in control hearts (up to 2%), approaching closely the zero level in the working myocardium of both the ventricles and interventricular septum, located at the considerable distance from the infarcted region. When similar experiments with ten-fold repeated³H-thymidine injections were performed between 15th and 20th post-infarction days the number of labeled myocyte nuclei was found to be reduced 4–6 times in atria, being changed rather a little in the perinecrotic ventricular myocardium and in the specialized myocardium of the atrioventricular system. Some possible reasons of the observed differences in the proliferative behaviour of cardiac myocytes in terms of their topology and/or specialization are discussed     

Variability of the cardiomyocyte ploidy in normal human hearts.

We have performed cytophotometry for DNA in isolated myocytes of the left ventricle from 16 men, aged 19-39 years, who died from various non-cardiac or pulmonary causes. The mean ploidy of myocytes varied from 3.2-3.9 c to 6.6-7.3 c in different layers of the anterior wall of the left ventricle (where c is the haploid DNA content measured by cytophotometry in Feulgen-stained preparations). There was no correlation between the layers. The percentage of binuclear cells varied from 25 to 86% and correlated in every layer with the mean ploidy value of the whole myocyte population. Approximate calculation of total ploidy revealed low values in the ventricles of some individuals, and high values in others. Averaging the values for all the hearts studied obscures this variation. Mean myocyte ploidy in different layers of the anterior wall was similar: in the external layer it was 5.1 +/- 0.3 c, in the middle layer 5.5 +/- 0.3 c and in the inner layer 4.8 +/- 0.4 c. The mean percentage of binuclear myocytes in these three layers was also similar, being 61 +/- 3%, 63 +/- 4% and 54 +/- 5%, respectively. Myocyte ploidy in tissue from the posterior wall of the left ventricle also varied, but was always higher than for the same layer of the anterior wall in the same ventricle. We propose that high or low myocyte ploidy, as well as different proportions of mono- and binucleate cells, can be a factor affecting the course and result of cardiac pathology in the absence of any changes of myocyte genome determined during early ontogenesis and representing a stable characteristic of the individual.     

Number and mass of the myocytes of the mouse heart

The mean number of cardiomyocytes is constant in the heart ventricles of 1, 1.5-2, 3-4, 5-6 and 12-month old mice. However, differences in the myocyte number among mice of the same age and with similar heart weight may reach 30%. There are only small differences in the mean ploidy among these mice. The mean protein content in the myocytes correlates to the ventricle weight. In some mice, however, no correlation was observed between the myocyte and ventricle weights, or between the calculated dry and wet weights of myocytes.     

Myocyte polyploidy in human cardiac pathology]

With general atherosclerosis, the ploidy of left ventricle myocytes in the hearts of patients that underwent infarction corresponds to the norm variation irrespective of the ventricle and heart weights. At heart diseases the myocyte nucleus ploidy is often much higher than the norm variability both in hypertrophied ventricles and in those with normal weight. An additional polyploidization is suggested that may occur at some natural ontogenetic periods of human development (in the childhood) during heart diseases both innate or spontaneously appearing at the particular time. Unlike, the myocardial hypertrophy in adults does not stimulate myocyte polyploidy.     

Effects of carbenoxolone on heart rhythm, contractility and intracellular calcium in streptozotocin-induced diabetic rat

Cardiac dysfunction is a frequently reported complication of clinical and experimental diabetes mellitus. Streptozotocin (STZ) – induced diabetes in rat is associated with a variety of cardiac defects including disturbances to heart rhythm and prolonged time-course of cardiac muscle contraction and/or relaxation. The effects of carbenoxolone (CBX), a selective gap junction inhibitor, on heart rhythm and contractility in STZ-induced diabetic rat have been investigated. Heart rate was significantly (P < 0.05) reduced in Langendorff perfused spontaneously beating diabetic rat heart (171±12 BPM) compared to age-matched controls (229± 9 BPM) and further reduced by 10⁻⁵ M CBX in diabetic (20%) and in control (17%) hearts. Action potential durations (APDs), recorded on the epicardial surface of the left ventricle, were prolonged in paced (6 Hz) diabetic compared to control hearts. Perfusion of hearts with CBX caused further prolongation of APDs and to a greater extent in control compared to diabetic heart. Percentage prolongation at 70% from the peak of the action potential amplitude after CBX was 18% in diabetic compared to 48% in control heart. CBX had no significant effect on resting cell length or amplitude of ventricular myocyte shortening in diabetic or control rats. However, resting fura-2 ratio (indicator for intracellular Ca²⁺ concentration) and amplitude of the Ca²⁺ transient were significantly (P < 0.05) reduced by CBX in diabetic rats but not in controls. In conclusion the larger effects of CBX on APD in control ventricle and the normalizing effects of CBX on intracellular Ca²⁺ in ventricular myocytes from diabetic rat suggest that there may be alterations in gap junction electrophysiology in STZ-induced diabetic rat heart.     

Cyclic strain induces proliferation of cultured embryonic heart cells

Summary Embryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% (P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% (P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain.     

Genome multiplication in cardiomyocytes of fast- and slow-growing mice

The number of myocytes and the percentage of cells with a high degree of ploidy increased in the heart ventricles of fast-growing mice compared with slow-growing ones. The mean incidence of octa- and hexadecaploid (by summary DNA content) myocytes was 7% in the slow-growing and 23% in the fast-growing, weaned mice. In these groups, the total myocyte number varied by 20%. There were 43% more myocyte genomes in the heart ventricles of the fast-growing mice than in those of the slow-growing mice. The same differences in cell number and ploidy persist in 90-day-old mice in spite of feeding ad libitum after weaning.     

Mechanobiology of cardiomyocyte development

Cardiac cells are under constant, self-generated mechanical stress which can affect the differentiation of stem cells into cardiac myocytes, the development of differentiated cells and the maturation of cells in neonatal mammals. In this article, the effects of direct stretch, electrically induced beating and substrate elasticity on the behavior and development of cardiomyocytes are reviewed, with particular emphasis on the effects of substrate stiffness on cardiomyocyte maturation. In order to relate these observations to in vivo mechanical conditions, we isolated the left ventricle of Black Swiss mice from embryonic day 13.5 through post-natal day 14 and measured the elastic modulus of the epicardium using atomic force microscope indentation. We found that the elastic modulus of the epicardium significantly changes at birth, from an embryonic value of 12±4 kPa to a neonatal value of 39±7 kPa. This change is in the range shown to significantly affect the development of neonatal cardiomyocytes.     

Telocytes and putative stem cells in ageing human heart

Tradition considers that mammalian heart consists of about 70% non‐myocytes (interstitial cells) and 30% cardiomyocytes (CMs). Anyway, the presence of telocytes (TCs) has been overlooked, since they were described in 2010 (visit www.telocytes.com ). Also, the number of cardiac stem cells (CSCs) has not accurately estimated in humans during ageing. We used electron microscopy to identify and estimate the number of cells in human atrial myocardium (appendages). Three age‐related groups were studied: newborns (17 days–1 year), children (6–17 years) and adults (34–60 years). Morphometry was performed on low‐magnification electron microscope images using computer‐assisted technology. We found that interstitial area gradually increases with age from 31.3 ± 4.9% in newborns to 41 ± 5.2% in adults. Also, the number of blood capillaries (per mm²) increased with several hundreds in children and adults versus newborns. CMs are the most numerous cells, representing 76% in newborns, 88% in children and 86% in adults. Images of CMs mitoses were seen in the 17‐day newborns. Interestingly, no lipofuscin granules were found in CMs of human newborns and children. The percentage of cells that occupy interstitium were (depending on age): endothelial cells 52–62%; vascular smooth muscle cells and pericytes 22–28%, Schwann cells with nerve endings 6–7%, fibroblasts 3–10%, macrophages 1–8%, TCs about 1% and stem cells less than 1%. We cannot confirm the popular belief that cardiac fibroblasts are the most prevalent cell type in the heart and account for about 20% of myocardial volume. Numerically, TCs represent a small fraction of human cardiac interstitial cells, but because of their extensive telopodes, they achieve a 3D network that, for instance, supports CSCs. The myocardial (very) low capability to regenerate may be explained by the number of CSCs, which decreases fivefold by age (from 0.5% to 0.1% in newborns versus adults).     

Growth of cardiac muscle cells during rat adaptation to altitude hypoxia

The weight of the right heart ventricle in 1.5-month-old rats kept after birth in the mountains of 3400 m altitude is higher and its muscle cell cytoplasm mass is much larger compared to those in 1.5-month-old animals raised at 800 m altitude. The hypertrophy of cells is not due to their polyploidization. Only a small increase in the relative number of polyploid cells takes place under high altitude hypoxia. The weight of the right ventricle and myocyte mass in 3-month-old rats kept 1.5-3 months after the birth at 3400 m altitude also increases, although this augmentation is significantly less than in the animals grown in the mountains for 1.5 months immediately after the birth. The myocyte ploidy of adult animals adapted to hypoxia does not essentially differ from that of 1.5- and 3-month-old control rats: about 80 per cent of these cells are polyploid. Thus, the growth of cardiac myocytes under the heart hyperfunction in the case of high altitude hypoxia proceeds mainly on the ground of the stable polyploid genome, as well as normal ontogenetic growth of these cells.     

A pharmaceutical preparation of Salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin II-stimulated collagen synthesis in fibroblasts

Salvia miltiorrhiza is a medicinal herb commonly used in traditional Chinese medicine for the prevention and treatment of cardiovascular disease. This study investigated the effects of Cardiotonic Pill (CP), a pharmaceutical preparation of Salvia miltiorrhiza, on cardiac myocytes and fibroblasts with respect to the viability, proliferation, and collagen synthesis in these cells under various conditions. A cardiac myocyte line, H9c2, and primarily cultured fibroblasts from rat hearts were incubated with CP over a broad concentration range (50–800 μg/ml) under normal cultures, conditions of ischemia (serum-free culture), and stimulation by angiotensin II (AII, 100 nM), hydrogen peroxide (H₂O₂, 50–200 μM), or tumor necrosis factor α (TNFα, 40 ng/ml) for 24–48 h. Cell growth, apoptosis, DNA and collagen synthesis, and expression of relevant genes were assessed via cell number study, morphological examination, Annexin-V staining, flow-cytometry, [³H]-thymidine or [³H]-proline incorporation assay, and Western blotting analysis. It was found that (1) at therapeutic (50 μg/ml) and double therapeutic (100 μg/ml) concentrations, CP did not significantly affect normal DNA synthesis and cell growth in these cardiac cells, while at higher (over 4-fold therapeutic) concentrations (200–800 μg/ml), CP decreased DNA synthesis and cell growth and increased cell death; (2) CP treatment (50 μg/ml) significantly inhibited TNFα-induced apoptosis in myocytes, with 12.3±1.46% cells being apoptosis in CP treatment group and 37.0±7.34% in the control (p<0.01), and simultaneously, expression of activated (phosphorylated) Akt protein was increased by about 2 folds in the CP-treated cells; and (3) in cultured fibroblasts, CP significantly reduced AII-induced collagen synthesis in a concentration-dependent manner (by ～50% and ～90% reduction of AII-induced collagen synthesis at 50 and 100 μg/ml, respectively). Thus, Salvia miltiorrhiza preparation CP is physiologically active on cardiac cells. The actions by CP to reduce apoptotic damage in myocytes and collagen synthesis in fibroblasts may help to preserve the heart function and reduce heart failure risk. The actions by CP to inhibit DNA synthesis and cell growth, which occurred at over therapeutic doses, may weaken the ability of heart repair. Further studies are needed to identify the chemical compounds in this herbal product that are responsible for these observed physiological effects.     

Cardiac myocytes' dynamic contractile behavior differs depending on heart segment

Cardiac myocytes originating from different parts of the heart exhibit varying morphology and ultrastructure. However, the difference in their dynamic behavior is unclear. We examined the contraction of cardiac myocytes originating from the apex, ventricle, and atrium, and found that their dynamic behavior, such as amplitude and frequency of contraction, differs depending on the heart segment of origin. Using video microscopy and high‐precision image correlation, we found that: (1) apex myocytes exhibited the highest contraction rate (～17 beats/min); (2) ventricular myocytes exhibited the highest contraction amplitude (～5.2 micron); and (3) as myocyte contraction synchronized, their frequency did not change significantly, but the amplitude of contraction increased in apex and ventricular myocytes. In addition, as myocyte cultures mature they formed contractile filaments, further emphasizing the difference in myocyte dynamics is persistent. These results suggest that the dynamic behavior (in addition to static properties) of myocytes is dependent on their segment of origin. Biotechnol. Bioeng. 2013; 110: 628–636. © 2012 Wiley Periodicals, Inc.     

Acute inhibitory effect of alpha‐mangostin on sarcoplasmic reticulum calcium‐ATPase and myocardial relaxation

The benefits of α‐mangostin for various tissues have been reported, but its effect on the heart has not been clarified. This study aimed to evaluate the effects of α‐mangostin on cardiac function. Using a cardiac sarcoplasmic reticulum (SR) membrane preparation, α‐mangostin inhibited SR Ca²⁺‐ATPase activity in a dose‐dependent manner (IC₅₀ of 6.47 ± 0.7 μM). Its suppressive effect was specific to SR Ca²⁺‐ATPase but not to myofibrillar Ca²⁺‐ATPase. Using isolated cardiomyocytes, 50 μM of α‐mangostin significantly increased the duration of cell relengthening and increased the duration of Ca²⁺ transient decay, suggesting altered myocyte relaxation. The relaxation effect of α‐mangostin was also supported in vivo after intravenous infusion. A significant suppression of both peak pressure and rate of ventricular relaxation (–dP/dt) relative to DMSO infusion was observed. The results from the present study demonstrated that α‐mangostin exerts specific inhibitory action on SR Ca²⁺‐ATPase activity, leading to myocardial relaxation dysfunction.     

Electromechanical coupling in cardiomyocytes from transmural layers of guinea pig left ventricle

The electrical and mechanical activity of heart ventricle cardiomyocytes is known to vary depending on the spatial location of cells in the wall, in particular, transmurally from the sub-endocardial layer to the sub-epicardial one. To investigate intracellular mechanisms of the functional heterogeneity of cardiomyocytes we developed mathematical models of the electromechanical coupling in cardiomyocytes from different transmural layers across the left ventricle (LV) wall of guinea pig. It is shown that the mechanisms of both direct linkages and feedback in the electromechanical coupling contribute to differences in both the shape and duration of action potential, and speed characteristics of contraction between isolated cardiac myocytes from the sub-endocardial and sub-epicardial layers.     

Analysis of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger (NCX) function and current in murine cardiac myocytes during heart failure

Na⁺/Ca²⁺ exchanger (NCX) plays important roles in cardiac electrical activity and calcium homeostasis. NCX current (I_NCX) shows transmural gradient across left ventricle in many species. Previous studies demonstrated that NCX expression was increased and transmural gradient of I_NCX was disrupted in failing heart, but the mechanisms underlying I_NCX remodeling still remain unknown. In present study, we used patch clamp technique to record I_NCX from subepicardial (EPI) myocytes and subendocardial (ENDO) myocytes isolated from sham operation (SO) mice and heart failure (HF) mice. Our results showed that I_NCX was higher in normal EPI cells compared with that in ENDO, whatever for forward mode or reverse mode. In HF group, I_NCX was significantly up-regulated, but EPI-ENDO difference was disrupted because of a more increase of I_NCX in ENDO myocytes. In order to explore the molecular mechanism underlying remodeling of I_NCX in failing heart, we detected the protein expression of NCX1 and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) by Western blot. We found that CaMKII activity was dramatically enhanced and parallel with the expression of NCX1 in failing heart. Our study demonstrated that transmural gradient of I_NCX existed in murine left ventricle, and increased activity of CaMKII should account for I_NCX remodeling in failing heart.     

A method to collect isolated myocytes and whole tissue from the same heart

A technique for isolation of cardiac myocytes and collection of whole heart tissue from individual hearts of adult rats is described in this study. After excision of the apical half of the left ventricle (LV) and cauterization of the cut edge, aortas were cannulated and high-quality isolated cardiac myocytes were collected after collagenase perfusion of the basal portion. Myocyte dimensions from the basal portion of cauterized and noncauterized hearts from matching rats were identical. Additionally, myocyte dimensions from the basal and apical halves of the LV were compared with the use of whole heart-isolated myocyte preps. No regional differences between basal and apical LV myocyte size were found. Therefore, this cauterization method can be used to collect isolated myocytes from the basal half and whole heart tissue from the apical half, with each half being representative of the other with respect to myocyte dimensions.     

Nuclear characteristics of cardiac myocytes following the proliferative response to mincing of the myocardium in the adult newt,Notophthalmus viridescens

Summary Amphibian cardiac myocytes are predominantly mononucleated and have been demonstrated to respond to injury with DNA synthesis and mitosis. The nature of this response with regard to nuclear number and ploidy is unclear. In this study, the apex of the newt ventricle was minced and replaced, increasing the reactive area of the wound. At 45 days after mincing following multiple injections of tritiated thymidine (2.5-Ci/animal, 20 Ci/mM) 15 to 20 days after mincing, three ventricular zones were isolated and fixed: Zone 1, the minced area; Zone 2, extending approximately 500 m proximally from the amputation plane; and Zone 3, the portion proximal to Zone 2. Myocytes separated in 50% KOH were examined for DNA synthesis by autoradiography and for nuclear number and DNA content using a scanning microdensitometer on Feulgen-Naphthol yellow S-stained cells. No labeled myocyte nuclei were found in control hearts and 98.3% of the myocytes were 2C. At 45 days, 46.78% of myocyte nuclei within Zone 1 were labeled, while 13% were non-diploid. In Zone 2, 9.25% were labeled with 4.8% non-diploid. In Zone 3, 1.1% were labeled, with 2.8% non-diploid. The newt ventricle's response to injury apparently may involve complete mitosis and cytokinesis, resulting in mononucleated diploid cells.     

Hypoxic postconditioning enhances the survival and inhibits apoptosis of cardiomyocytes following reoxygenation: role of peroxynitrite formation

Objectives: Our previous study has shown that slow or “controlled” reperfusion for the ischemic heart reduces cardiomyocyte injury and myocardial infarction, while the mechanisms involved are largely unclear. In this study, we tested the hypothesis that enhancement of survival and prevention of apoptosis in hypoxic/reoxygenated cardiomyocytes by hypoxic postconditioning (HPC) are associated with the reduction in peroxynitrite (ONOO⁻) formation induced by hypoxia/reoxygenation (H/R). Methods: Isolated adult rat cardiomyocytes were exposed to 2 h of hypoxia followed by 3 h of reoxygenation. After 2 h of hypoxia the cardiomyocytes were either abruptly reperfused with pre-oxygenized culture medium or postconditioned by two cycles of 5 min of brief reoxygenation and 5 min of re-hypoxia followed by 160 min of abrupt reoxygenation. Results: H/R resulted in severe injury in cardiomyocytes as evidenced by decreased cell viability, increased LDH leakage in the culture medium, increased apoptotic index (P values all less than 0.01 vs. normoxia control group) and DNA ladder formation, which could be significantly attenuated by HPC treatment applied before the abrupt reoxygenation (P < 0.05 vs. H/R group). In addition, H/R induced a significant increase in ONOO⁻ formation as determined by nitrotyrosine content in cardiomyocytes (P < 0.01 vs. normoxia control). Treatment with the potent ONOO⁻ scavenger uric acid (UA) at reoxygenation significantly decreased ONOO⁻ production and protected myocytes against H/R injury, whereas the same treatment with UA could not further enhance myocyte survival in HPC group (P > 0.05 vs. HPC alone). Statistical analysis showed that cell viability closely correlated inversely with myocyte ONOO⁻ formation (P < 0.01). Conclusion: These data demonstrate that hypoxic postconditioning protects myocytes against apoptosis following reoxygenation and enhances myocytes survival, which is partly attributable to the reduced ONOO⁻ formation following reoxygenation. H.-C. Wang and H.-F. Zhang contributed equally to this study.