Resistance to osimertinib in advanced EGFR-mutated NSCLC: a prospective study of molecular genotyping on tissue and liquid biopsies.

BACKGROUND: Resistance to osimertinib in advanced EGFR-mutated non-small cell lung cancer (NSCLC) constitutes a significant challenge for clinicians either in terms of molecular diagnosis and subsequent therapeutic implications. METHODS: This is a prospective single-centre study with the primary objective of characterising resistance mechanisms to osimertinib in advanced EGFR-mutated NSCLC patients treated both in first- and in second-line. Next-Generation Sequencing analysis was conducted on paired tissue biopsies and plasma samples. A concordance analysis between tissue and plasma was performed. RESULTS: Sixty-five advanced EGFR-mutated NSCLC patients treated with osimertinib in first- (n = 56) or in second-line (n = 9) were included. We managed to perform tissue and liquid biopsies in 65.5% and 89.7% of patients who experienced osimertinib progression, respectively. Acquired resistance mechanisms were identified in 80% of 25 patients with post-progression samples, with MET amplification (n = 8), EGFR C797S (n = 3), and SCLC transformation (n = 2) the most frequently identified. The mean concordance rates between tissue and plasma for the EGFR activating mutation and for the molecular resistance mechanisms were 87.5% and 22.7%, respectively. CONCLUSIONS: Resistance to osimertinib demonstrated to be highly heterogeneous, with MET amplification the main mechanism. Plasma genotyping is a relevant complementary tool which might integrate tissue analysis for the study of resistance mechanisms.

Comparison of HER2 status in primary and paired metastatic sites of gastric carcinoma.

BACKGROUND: Trastuzumab has recently shown efficacy in the treatment of HER2-positive advanced gastric adenocarcinoma. Although antibody-based therapies target the metastatic disease, HER2 status is usually evaluated in the primary tumour because metastatic sites are rarely biopsied. The aim of this study was to compare HER2 status in primary and paired metastatic sites of gastric adenocarcinoma. METHODS: The HER2 status was assessed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in 72 secondary lesions of gastric adenocarcinoma and in the corresponding primary tumours. RESULTS: Concordance of FISH results, evaluable in 68 primary and matched metastatic sites, was 98.5%. Concordance of IHC results, available in 39 of the 72 paired cases, was 94.9%. Only one case showed discordance between primary tumour and metastasis, being negative by both IHC and FISH in the primary and showing HER2 overexpression and amplification in the corresponding pancreatic lymph node metastasis. CONCLUSION: The high concordance observed between HER2 results obtained by both IHC and FISH on primary tumours and corresponding metastases suggests that in gastric cancer HER2 status is maintained in most cases unchanged during the metastatic process.

PTEN status in advanced colorectal cancer treated with cetuximab.

BACKGROUND: Loss of phosphatase and tensin homologue deleted in chromosome 10 (PTEN) function in advanced colorectal cancer (CRC) may represent one of the resistance mechanisms to cetuximab by interfering with the epidermal growth factor receptor signal transduction pathway. METHODS: PTEN expression tested by indirect immunofluorescence was evaluated both on primary (n=43) and on metastatic (n=24) sites in CRC patients treated with cetuximab. RESULTS: The loss of PTEN expression tested on metastatic sites was negatively associated with response (100% progressive disease (PD) in PTEN-negative cases vs 30% PD in PTEN-positive cases; P<0.05), PFS (0.8 vs 8.2 months; P<0.001) and OS (2.9 vs 14.2 months; P<0.001). CONCLUSION: A potential role of PTEN in the anti-tumour activity of cetuximab could be hypothesised.

Gender differences and aging: effects on the human heart.

OBJECTIVES: This study investigated the changes in myocyte size and number in the left and right ventricles that occur with aging in the female and male heart. BACKGROUND: Differences in life span between women and men may be related to a better preservation of myocardial structure in the female heart with aging. On this basis, the hypothesis was advanced that the aging process has a different impact on the integrity of the myocardium in the two genders. METHODS: Morphometric methodologies were applied to analyze the changes in number and size of ventricular myocytes in the hearts of 53 women and 53 men. The changes in mononucleated and binucleated myocytes with age were determined in enzymatically dissociated cells. The age interval examined varied from 17 to 95 years. RESULTS: Aging was associated with a preservation of ventricular myocardial mass, aggregate number of mononucleated and binucleated myocytes, average cell diameter and volume in the female heart. In contrast, nearly 1 g/year of myocardium was lost in the male heart, and this phenomenon accounted for the loss of approximately 64 million cells. This detrimental effect involved the left and right sides of the heart. In the remaining cells, myocyte cell volume increased at a rate of 158 microns3/year in the left and 167 microns3/year in the right ventricle. CONCLUSIONS: Aging does not lead to myocyte cell loss and myocyte cellular reactive hypertrophy in women, indicating that gender differences may play a significant role in the detrimental effects of the aging process on the heart.

Effects of genetic hypertension and nutritional anaemia on ventricular remodelling and myocardial damage in rats.

OBJECTIVE: In order to determine whether alterations in cardiac function and structure occur early in life in spontaneously hypertensive rats (SHR) and whether the addition of a volume load would affect myocardial growth and haemodynamic performance, SHR were exposed to an iron and copper deficient diet for 12 weeks (SHR-A) and compared with untreated SHR and Wistar Kyoto controls (WKY). RESULTS: Systolic arterial blood pressure increased in SHR, whereas nutritional anaemia prevented the rise of blood pressure in SHR-A. The diet employed provoked a severe hypochromic microcytic anaemia with a marked reduction in blood viscosity and increased volume load on the heart in SHR-A. Genetically determined hypertension alone induced a 16% increase in left ventricular weight and an increase in left ventricular peak systolic pressure (LVPSP) and +dP/dt. The superimposition of anaemia resulted in a 43% expansion in left ventricular weight with a decrease in LVPSP and +dP/dt, and an increase in left ventricular end diastolic pressure. Wall thickening and a preservation of chamber volume occurred in SHR, while SHR-A had a degree of ventricular dilatation which exceeded the extent of wall thickening. However, genetic hypertension was accompanied by myocardial tissue injury which was fully prevented by the addition of nutritional anaemia. Moreover, the capillary volume was decreased in SHR and increased in SHR-A. CONCLUSIONS: Genetically determined hypertension in combination with anaemia results in eccentric ventricular hypertrophy and cardiac dysfunction in spite of an increase in capillary luminal volume and limited structural damage.

Dependence of temporal variability of ventricular recovery on myocardial fibrosis. Role of mechanoelectric feedback?

OBJECTIVE: The study was aimed at establishing the effect of factors involved in the expression of mechanoelectric feedback in the heart, such as R-R interval and connective tissue, on time dependent changes in ventricular recovery, as determined at the body surface by beat to beat variability of QRST integral maps (BBV-IM). METHODS: We used 15 normal 6-month-old Wistar rats. In each anesthetized animal, we performed a 3-minute continuous recording of 44. The simultaneous chest ECGs. The signals were interactively processed, 1) to determine mean R-R interval and R-R variability throughout the recording period and 2) to compute QRST integral maps from approximately 50 beats belonging to the end of expiration. Then BBV-IM was calculated and expressed as percentage of beats significantly differing from a template. At sacrifice, the amount of myocardial fibrosis was morphometrically evaluated. RESULTS: R-R interval was 149 ms +/- 4, R-R interval variability 0.008 +/- 0.001 and BBV-IM 30.7% +/- 4.4. Myocardial fibrosis expressed as % volume of left ventricular myocardium, numerical density of fibrotic foci and average cross-sectional area of the foci was 3.0% +/- 0.4, 3.8 +/- 0.6 and 4.4 microns(2)/1000 +/- 0.1 respectively, BB-IM was positively correlated to the % volume of fibrosis (r = 0.83, P < 0.0003). Both measurements were positively correlated to R-R interval (BBV-IM: r = 0.83, P < 0.0001; % volume of fibrosis: r = 0.87, P < 0.001) and negatively correlated to cardiac weights (BBV-IM: r = -0.79, P < 0.0005; % volume of fibrosis: r = -0.75, P < 0.001). CONCLUSION: Beat to beat changes in ventricular repolarization attributable to mechanoelectric transduction can be detected at the body surface by means of BBV-IM.

Enalapril prevents cardiac fibrosis and arrhythmias in hypertensive rats.

To evaluate the effects of hypertension on cardiac hypertrophy, on myocardial structure, and on ventricular arrhythmias, 27 3-month-old spontaneously hypertensive rats were treated with enalapril (10 mg/kg) daily for 11 months and compared with 26 untreated control rats. Systolic arterial pressure was significantly decreased in treated rats, and at the end of the experiment, it was 199 +/- 3 mm Hg (treated) versus 237 +/- 3 mm Hg (controls) (p less than 0.001). At this time, spontaneous arrhythmias and induced arrhythmias either by programmed electrical stimulation (train of stimuli +1 or 2 extrastimuli) or by trains of eight stimuli at decreasing coupling intervals were observed in isolated heart preparations. Comparing enalapril-treated and control rats, spontaneous arrhythmias (9 of 27 versus 20 of 26, respectively; p less than 0.01), programmed stimulation-induced arrhythmias (3 of 26 versus 12 of 23, respectively; p less than 0.01), and trains of stimuli-induced arrhythmias (4 of 26 versus 14 of 19, respectively, p less than 0.001) were less frequent in the enalapril group. Left ventricular weight was decreased in treated rats by 18% (p less than 0.001). Enalapril administration diminished the fraction of myocardium occupied by foci of replacement fibrosis normally occurring in control rats by 59% (p less than 0.001). Finally, a significant correlation was found between left ventricular weight, the extent of myocardial fibrosis, and the occurrence of ventricular fibrillation. It was concluded that chronic treatment with enalapril, which resulted in attenuation of systemic arterial pressure by limiting cardiac hypertrophy and myocardial fibrosis, decreases the propensity of the heart of hypertensive rats to arrhythmogenesis.

Motor responses of rat hypertrophic intestine following chronic obstruction.

The present work aims at investigating the changes in motor responsiveness of rat intestine hypertrophied by chronic mechanical obstruction. Motor responses to pharmacological agents and electrical field stimulation (EFS) were studied in hypertrophic ileal segments excised from rats subjected to experimental stenosis (n = 20) and compared with responses of control tissues from sham-operated animals (n = 20). Spontaneous motility and contractile responses to exogenous agents (KCl, acetylcholine and substance P) and EFS (10-s trains every minute, 120 mA, 0.5 ms, 1-10 Hz) were increased in hypertrophic longitudinal segments; however, normalization of motor responses to tissue wet weight revealed a remarkable reduction of contractile efficiency in hypertrophied tissues coupled with a loss of sensitivity to nitric oxide-mediated relaxation. Furthermore, EFS under non-adrenergic non-cholinergic (NANC) conditions unveiled a major role of the cholinergic component over the peptidergic one in the neurogenic contraction of hypertrophic intestine. On the whole, hypertrophic intestinal growth emerges as a dynamic process entailing adaptation of smooth muscle and neuronal structures to the increased functional load imposed by lumen obstruction.

Development and validation of an high performance liquid chromatography-tandem mass spectrometry method for the determination of imatinib in rat tissues.

An high performance liquid chromatography-tandem mass-spectrometry (HPLC-MS/MS) method was developed and validated for the determination in rat heart and liver of the tyrosine kinase inhibitor imatinib (IM), an anticancer drug approved for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Extraction of the drug from tissues was performed by solvent extraction and the obtained extracts were analyzed by HPLC-MS/MS in selected reaction monitoring mode. The developed method was validated according to the criteria for bioanalytical method, showing good performances in terms of lower limit of quantification (LLOQ=0.02µgml(-1)), linearity (R(2)=0.998), repeatability (RSD<3%), reproducibility (RSD<13%) and recovery (RR>89%). The developed method was then applied to the analysis of heart and liver of rats treated with different doses of IM, with and without the simultaneous administration of carvedilol, a beta-blocking agent with cardioprotective effect, in order to evaluate tissue levels of the tyrosine kinase inhibitor. The obtained results revealed that the amount of IM in the rat heart was significantly affected by the administered dose, whereas carvedilol had no effect on IM concentrations. Thus, we have developed a method that allows the detection of IM traces in complex tissues such as the heart and liver and that may be proposed for the determination of the drug in other clinically relevant biological samples.

Resident cardiac stem cells.

The introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathologic heart and opens new options for the treatment of cardiovascular diseases. The feasibility of adult bone marrow autologous and allogenic cell therapy of ischemic cardiomyopathies has been demonstrated in humans. However, many unresolved questions remain to link experimental with clinical observations. The demonstration that the heart is a self-renewing organ and that its cell turnover is regulated by myocardial progenitor cells offers novel pathogenetic mechanisms underlying cardiac diseases and raises the possibility to regenerate the damaged heart. Indeed, cardiac stem progenitor cells (CSPCs) have recently been isolated from the human heart by several laboratories although differences in methodology and phenotypic profile have been described. The present review points to the potential role of CSPCs in the onset and development of congestive heart failure and its reversal by regenerative approaches aimed at the preservation and expansion of the resident pool of progenitors.

Effect of long-term administration of two beta-blocking agents on myocardial structure in spontaneously hypertensive rats.

Two vasodilating beta-blockers, dilevalol and pindolol, were studied for their effects on the development of hypertension and left ventricular hypertrophy in spontaneously hypertensive rats. Dilevalol has agonist activity at beta 2-, whereas pindolol interacts with both beta 1- and beta 2-receptor subtypes. Groups of 7-week old spontaneously hypertensive rats were given dilevalol (30 mg/kg) or pindolol (3 mg/kg) in the drinking water for 3 months. A control group of spontaneously hypertensive rats and age-matched normotensive Wistar Kyoto rats were also used. Systolic blood pressure and heart rate were recorded weekly. Cardiac structural changes were determined by morphometric analysis. Control spontaneously hypertensive rats developed hypertension and left ventricular hypertrophy as compared with normotensive rats. Dilevalol and pindolol prevented the rise in blood pressure, but only dilevalol lowered heart rate. However, neither of the compounds prevented the development of left ventricular hypertrophy, but they differently influenced myocardial structure as measured by the volume fraction of fibrotic tissue in the left ventricle. Dilevalol markedly prevented the amount of fibrosis to a level comparable to that found in normotensive rats, whereas pindolol had a weaker effect. The data suggest that individual pharmacologic characteristics, such as partial agonist activity of each beta-blocker examined, may account for differential effects on the myocardial structure.

Long-term pressure-induced cardiac hypertrophy: capillary and mast cell proliferation.

To determine whether a prolonged duration of mechanical load on the ventricular myocardium leads to capillary proliferation, constriction of the pulmonary artery was produced in rats at 3 mo of age, and the hearts were examined 200 days later. Functionally, elevations of right ventricular systolic pressure, from 30 +/- 12 to 67 +/- 11 mmHg, and right ventricular end-diastolic pressure, from 2.4 +/- 1.4 to 8.6 +/- 2.4 mmHg, were found. Anatomically, a 69% enlargement in right ventricular mass was observed, and this hypertrophic growth was characterized by a 78% thickening of the wall with no change in ventricular wall area, the latter calculated from the quotient of ventricular volume and wall thickness. Morphometric data showed a 44% increase in the capillary-to-myocyte ratio, a 66% augmentation of the total length of capillaries in the whole ventricle, and a 77% greater number of capillaries across the ventricular wall. Furthermore, these indexes of neogenesis of capillary units were associated with a more than three fold increase in the numerical density of mast cell profiles in the myocardium. In conclusion, capillary proliferation occurs in the adult rat heart, and this process appears to be coupled with mast cell hyperplasia.

Cellular basis of ventricular remodeling after myocardial infarction in rats.

The remodeling of the spared non-ischemic left ventricular myocardium after different time intervals from the occlusion of the left coronary artery was examined in rats. In the presence of large infarcts, ventricular failure developed two to three days after surgery, because of chamber dilation and thinning of the wall, resulting in an average 7.5-fold increase in diastolic stress on the surviving myocardium. Mural thinning of the ventricular wall remote from and bordering the infarction occurred through side-to-side slippage of myocytes and capillaries within the wall. Although an average hypertrophic growth of 22% of the spared myocytes has been found, this amount of hypertrophy was insufficient to restore normal myocardial function. Long-term cardiac restructuring after infarction was characterized by the persistence of chamber dilatation and thinning of the ventricular wall. In addition to the side-to-side slippage, lengthening of the myocytes was an important cause of ventricular changes. As the reactive hypertrophy of the unaffected ventricle was insufficient to re-establish the ratio of ventricular mass to chamber volume, the diastolic stress remained elevated and decompensated eccentric ventricular hypertrophy developed. The anatomical remodeling of the spared left ventricular myocardium is an important conditioning factor in the short- and long-term outcome of ischemic cardiomyopathy.

Effects of an early treatment with lisinopril and isosorbide-5-mononitrate on hemodynamics and late ventricular remodelling in rats with 9-week myocardial infarction.

This study was undertaken to assess whether the converting enzyme inhibitor lisinopril, and the long-acting nitrate, isosorbide-5-mononitrate, affect left ventricle dysfunction and anatomical remodelling in rats with myocardial infarction. Lisinopril, isosorbide-5-mononitrate or vehicle were given to rats (n = 10-14 per group) immediately after coronary artery occlusion (by an intravenous bolus) and then for nine weeks (in drinking water). At the end of the study, left ventricular pressures were measured, the heart arrested in diastole, and infarct size, left ventricular chamber volume and wall thicknesses measured. Lisinopril significantly lowered systemic blood pressure and left ventricular systolic pressure in rats with small (< 15% scarred tissue of the left ventricle) and large (> 15%) infarcts; the weight of the left ventricle (including the septum) was reduced by 24% and 28% in animals with small and large infarcts, respectively. Lisinopril lowered left ventricular end-diastolic pressure (by 33% and 39%) and chamber volume (by 4% and 34%) in rats with small and large infarcts, respectively, compared with controls (NS). The combined anatomical and hemodynamic changes led to a reduction of the circumferential wall stress by 20% and 44% in lisinopril-treated rats with small and large infarcts, respectively (NS). No significant changes were seen in the nitrate-treated hearts compared with controls. Lisinopril, given early after myocardial infarction and continued for nine weeks, significantly affected cardiac hemodynamics and ventricular weights in rats with infarcts of different sizes.(ABSTRACT TRUNCATED AT 250 WORDS)

Spirapril prevents left ventricular hypertrophy, decreases myocardial damage and promotes angiogenesis in spontaneously hypertensive rats.

To test whether angiotensin-converting enzyme (ACE) inhibition may prevent myocardial damage and may affect coronary microvasculature in spontaneously hypertensive rats (SHR), young 5-week-old SHR were treated for 3 months with spirapril and changes in blood pressure (BP) were monitored. Untreated SHR were used as controls. The rats were killed; left ventricular (LV) shape, weight, and wall thickness were examined and the ventricular myocardium was analyzed morphometrically to determine the effect of the drug on the relative amount, number per unit area of myocardium, and average dimension of foci of myocardial scarring. Moreover, volume fraction, surface, numerical density, and diffusion distance for oxygen of the coronary capillaries were analyzed. BP remained 20-30% lower in treated SHR with respect to controls, and LV weight and thickness decreased 20 and 21%, respectively. The number and dimension of the foci of fibrosis were reduced, resulting in an overall 68% decrement in the amount of myocardial damage. Finally, a 28% increment in numerical density of capillary profiles associated with a 13% reduction in their cross-sectional area decreased the diffusion distance for oxygen from the capillary wall to the myocytes by 14% in treated SHR. Spirapril decreases BP and LV weight and thickness in the SHR model of hypertension and substantially improves coronary capillary microvasculature, decreasing hypertensive myocardial damage. These results may be attributed to inhibition of the systemic effects of angiotensin II (AII) as well as to a local protective action of the drug against possible intramyocardial AII production.

Cellular basis of wall remodeling in long-term pressure overload-induced right ventricular hypertrophy in rats.

To determine the effects of long-term pressure overload on the structural mechanisms implicated in wall remodeling of the right ventricle, a mild pulmonary artery banding was applied to rats approximately 2 months old, and the animals were killed 150 days later. The surgical procedure resulted in a 60% reduction in the cross-sectional area of the constricted vessel and a 52% increase in the weight of the right ventricle. The hypertrophic myocardial response was associated with an elevation in right ventricular systolic pressure (from 33 +/- 11 mm Hg to 71 +/- 12 mm Hg), right ventricular end-diastolic pressure (from 3 +/- 1 mm Hg to 10 +/- 3 mm Hg), and central venous pressure (from 2 +/- 0.2 mm Hg to 10 +/- 3 mm Hg). The 76% increase in wall thickness after pulmonary artery stenosis was the result of a 24% lateral expansion of cardiac muscle cells and a 44% increase in the number of myocytes across the ventricular wall. The intermyocyte distance was also increased by 22%. These cellular adaptations occurred with no alterations in total myocyte length, average sarcomere length, and volume composition of the myocardium. Ventricular wall area was decreased by 14%, which suggests a small reduction in chamber volume. Myocyte growth was accompanied by proportional expansions of mitochondrial and myofibrillar components, so that the ratio of mitochondria to myofibrils in the cytoplasm remained essentially constant. In conclusion, ventricular remodeling in this model of chronic pressure hypertrophy is characterized by increases in cellular diameter and number that would both tend to decrease the magnitude of systolic and diastolic stresses on a per cell basis and thus improve the myocardial response to a prolonged and sustained mechanical load.

Capillary growth in anemia-induced ventricular wall remodeling in the rat heart.

To determine whether anemia-induced cardiac hypertrophy affects ventricular size and shape and the component structures of the capillary network of the left and right ventricles, young male rats were fed an iron- and copper-deficient diet for 7 weeks. By that time, blood hemoglobin content fell to 5 +/- 1 g/dl, and packed cell volume fell to 18 +/- 3%. To further characterize the implications of anemia, red blood cell number, hemoglobin corpuscular content, systemic arterial pressure, heart rate, and blood viscosity were measured. Moreover, the changes in ventricular weights were analyzed in terms of the alterations in ventricular wall area and ventricular wall thickness to establish the impact of the elevation in load associated with a high cardiac output state on ventricular remodeling. The quantitative properties of the capillary circulation were also examined biventricularly by low power electron microscopic morphometry to evaluate the adaptive growth potential of the coronary microcirculation in this form of cardiac hypertrophy. Anemia was found to interfere with the production of red blood cells and their mean corpuscular hemoglobin content and resulted in a 40% reduction in blood viscosity and a 12% and 27% decrease in systolic and diastolic blood pressure, respectively. The changes in heart rate were not statistically significant. In comparison with control animals, heart weight increased by 50%, but the enlargement in right ventricular mass (65%) was greater than that of the left ventricle (47%). Ventricular hypertrophy occurred with increases in wall area and wall thickness although the former increased consistently more than the latter in either ventricle. Tissue growth was accompanied by a 60% lengthening of the capillary network, which in combination with an increase in capillary diameter resulted in a 65% and 34% expansion in capillary luminal volume and 56% and 20% larger luminal surface density in the left and right sides of the heart, respectively. In conclusion, hypochromic microcytic anemia leads to eccentric ventricular hypertrophy with a significant amount of capillary proliferation that may tend to protect the myocardium from the increased potential for ischemic injury.

Myocyte cellular hypertrophy and hyperplasia contribute to ventricular wall remodeling in anemia-induced cardiac hypertrophy in rats.

To determine the effects of chronic anemia on the functional and structural characteristics of the heart, 1-month-old male rats were fed a diet deficient in iron and copper, which led to a hemoglobin concentration of 4.63 g/dl, for 8 weeks. At sacrifice, under fentanyl citrate and droperidol anesthesia, systolic, diastolic, and mean arterial blood pressures were decreased, whereas differential pressure was increased. Left ventricular systolic pressure and the ventricular rate of pressure rise (mmHg/s) were reduced by 9% and 14%, respectively. Moreover, developed peak systolic ventricular pressure and maximal dP/dt diminished 14% and 12%. After perfusion fixation of the coronary vasculature and the myocardium, at a left ventricular intracavitary pressure equal to the in vivo measured end diastolic pressure, a 10% thickening of the left ventricular wall was measured in association with a 13% increase in the equatorial cavitary diameter and a 44% augmentation in ventricular mass. The 52% hypertrophy of the right ventricle was characterized by an 11% thicker wall and a 37% larger ventricular area. The 33% expansion in the aggregate myocyte volume of the left ventricle was found to be due to a 14% myocyte cellular hypertrophy and a 17% myocyte cellular hyperplasia. These cellular parameters were calculated from the estimation of the number of myocyte nuclei per unit volume of myocardium in situ and the evaluation of the distribution of nuclei per cell in enzymatically dissociated myocytes. Myocyte cellular hyperplasia provoked a 9% increase in the absolute number of cells across the left ventricular wall. In contrast, myocyte cellular hypertrophy (42%) was responsible for the increase in myocyte volume of the right ventricle. The proliferative response of left ventricular myocytes was not capable of restoring diastolic cell stress, which was enhanced by the changes in ventricular anatomy with anemia. In conclusion, chronic anemia induced an unbalanced load on the left ventricle, which evoked a hyperplastic reaction of preexisting myocytes, in an attempt to normalize diastolic wall and myocyte stress.