The adult heart responds to increased workload with physiologic hypertrophy, cardiac stem cell activation, and new myocyte formation.

AIMS: It is a dogma of cardiovascular pathophysiology that the increased cardiac mass in response to increased workload is produced by the hypertrophy of the pre-existing myocytes. The role, if any, of adult-resident endogenous cardiac stem/progenitor cells (eCSCs) and new cardiomyocyte formation in physiological cardiac remodelling remains unexplored. METHODS AND RESULTS: In response to regular, intensity-controlled exercise training, adult rats respond with hypertrophy of the pre-existing myocytes. In addition, a significant number (∼7%) of smaller newly formed BrdU-positive cardiomyocytes are produced by the exercised animals. Capillary density significantly increased in exercised animals, balancing cardiomyogenesis with neo-angiogenesis. c-kit(pos) eCSCs increased their number and activated state in exercising vs. sedentary animals. c-kit(pos) eCSCs in exercised hearts showed an increased expression of transcription factors, indicative of their commitment to either the cardiomyocyte (Nkx2.5(pos)) or capillary (Ets-1(pos)) lineages. These adaptations were dependent on exercise duration and intensity. Insulin-like growth factor-1, transforming growth factor-ß1, neuregulin-1, bone morphogenetic protein-10, and periostin were significantly up-regulated in cardiomyocytes of exercised vs. sedentary animals. These factors differentially stimulated c-kit(pos) eCSC proliferation and commitment in vitro, pointing to a similar role in vivo. CONCLUSION: Intensity-controlled exercise training initiates myocardial remodelling through increased cardiomyocyte growth factor expression leading to cardiomyocyte hypertrophy and to activation and ensuing differentiation of c-kit(pos) eCSCs. This leads to the generation of new myocardial cells. These findings highlight the endogenous regenerative capacity of the adult heart, represented by the eCSCs, and the fact that the physiological cardiac adaptation to exercise stress is a combination of cardiomyocyte hypertrophy and hyperplasia (cardiomyocytes and capillaries).

Influence of age, sex, and aerobic capacity on forearm and skin blood flow and vascular conductance.

This study investigated the influence of age, sex, and aerobic capacity on resting and peak forearm and cutaneous blood flow (FBF, CBF). We recruited 93 female and 129 male subjects (age range 16-76 years). FBF and CBF were assessed by plethysmography and laser-Doppler flowmetry, respectively. Peak FBF was obtained following 5 min forearm vascular occlusion and peak CBF in response to local skin heating of 42 degrees C. Blood pressure was measured manually and by Finapres. Maximal oxygen uptake (VO2max) was obtained from a treadmill exercise stress test. Age was associated with declines in resting FBF (y = -1.176 ln(x) + 6.6899, r(2) = 0.45) and peak FBF (y = -17.21 ln(x) + 93.843, r(2) = 0.53) (both p < 0.05). Peak CBF decreased with increasing age (y = -223.6 ln(x) + 1,102.9, r(2) = 0.34) (p < 0.05), but resting CBF was unchanged (p > 0.05). Males had higher resting and peak FBF than females (p < 0.05) and these variables were related to ageing better in males (y = -1.245 ln(x) + 7.188, r(2) = 0.71 and y = -18.53 ln(x) + 102.82, r(2) = 0.69) than in females (y = -1.149 ln(x) + 6.4307, r(2) = 0.38 and y = -16.59 ln(x) + 88.872, r(2) = 0.55). There were no sex differences in resting CBF (p > 0.05). Peak CBF was much better related to ageing in males than females (y = -276.1 ln(x) + 1,365.4, r(2) = 0.53 vs. y = -183.1 ln(x) + 907.86, r(2) = 0.28). VO2max decreased with advancing age and this decline was associated with the decline in peak FBF (y = -0.5933x + 10.91, r(2) = 0.36, p < 0.05) but not with peak CBF (p > 0.05). These results suggest that healthy ageing is associated with a curvilinear decline in resting and peak forearm and peak cutaneous vasodilator capacity, with males more severely affected than females. The data indicate that peak FBF is influenced by VO2max but peak CBF is not.

The effect of 48 weeks of aerobic exercise training on cutaneous vasodilator function in post-menopausal females.

Skin blood flow (SkBF) and endothelial-dependent vasodilatation decline with ageing and can be reversed with exercise training. We tested whether 48 weeks of training could improve SkBF and endothelial function in post-menopausal females; 20 post-menopausal subjects completed the study. SkBF was measured by laser-Doppler flowmetry (LDF). Cutaneous vascular conductance (CVC) was calculated as LDF/blood pressure. Resting CVC was measured at 32 degrees C and peak CVC at 42 degrees C. Cutaneous endothelial-dependent and -independent vasodilatations were determined by the iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. All assessments described were performed at entry (week 0), and after 6, 12, 24, 36, and 48 weeks of training. Resting CVC measures did not change (P > 0.05) throughout the study. Peak CVC increased (P < 0.05) after 24 weeks (7.2 +/- 1.2 vs. 11.6 +/- 1.4 AU mmHg(-1)) and at the 36- and 48-week assessments (13.0 +/- 1.7 and 14.9 +/- 2.1 AU mmHg(-1), respectively). Responses to ACh also increased (P < 0.05) at the 24-week assessment (5.1 +/- 2.1 vs. 8.55 +/- 2.3 AU mmHg(-1)) and increased further at the 36 and 48-week assessments (11.6 +/- 3.7 and 13.2 +/- 3.9 AU mmHg(-1), respectively). Cutaneous responses to SNP increased (P < 0.05) after 36 weeks (8.7 +/- 2.1 vs. 13.02 +/- 2.23 AU mmHg(-1) at 36 weeks). VO(2max) increased after 12 weeks (23.5 +/- 0.7 vs. 25.4 +/- 0.9 ml kg(-1) min(-1)) and improved (P < 0.05) further throughout the study (31.6 +/- 1.8 ml kg(-1) min(-1) at week 48). Aerobic exercise produces positive adaptations in the cutaneous vasodilator function to local heating as well as in cutaneous endothelial and endothelial-independent vasodilator mechanisms. Aerobic capacity was also significantly improved. These adaptations were further enhanced with progressive increases in exercise intensity.

Proteomic investigation of changes in human vastus lateralis muscle in response to interval-exercise training.

No previous study has used proteomics to investigate the effects of exercise training on human skeletal muscle. Five recreationally active men completed a 6-wk training programme involving three sessions per week, utilising six 1-min bouts at maximum oxygen uptake (V O(2)max) interspersed with 4 min at 50% V O(2)max. Vastus lateralis was biopsied at standardised times before and after the training intervention. Protein expression profiling was performed using differential analysis of 2-DE gels; complemented with quantitative analysis (iTRAQ) of tryptic peptides from 1-DE gel lane-segments using LC-MALDI MS/MS. Interval training increased average V O(2)max (7%; p<0.001) and was associated with greater expression of mitochondrial components, including succinate dehydrogenase, trifunctional protein-alpha and ATP synthase alpha- and beta-chains. 2-DE resolved 256 spots, and paired t-tests identified 20 significant differences in expression (false discovery rate <10%). Each differentially expressed gene product was present as multiple isoelectric species. Therefore, the differences in spot expression represent changes in post-transcriptional or post-translational processing. In particular, modulation of muscle creatine kinase and troponin T were prominent. Pro-Q Diamond staining revealed these changes in expression were associated with phosphorylated protein species, which provides novel information regarding muscle adaptation to interval training.

Maximizing patient benefit from cardiac resynchronization therapy with the addition of structured exercise training: a randomized controlled study.

OBJECTIVES: We evaluated the benefits of additional exercise training after cardiac resynchronization therapy (CRT). BACKGROUND: Cardiac resynchronization therapy results in improved morbidity and mortality in appropriate patients. We hypothesized that a structured exercise training program in addition to CRT would maximize the improvements in exercise capacity, symptoms, and quality of life (QOL). METHODS: Fifty patients referred for CRT were recruited. Patients were assessed before and 3 and 6 months after CRT. Functional class and QOL scores were recorded, and exercise tests were performed with hemodynamic measurements. Peak lower limb skeletal muscle torque was measured during extension, and echocardiography was undertaken at each visit. At 3 months, patients were randomized with a simple sealed envelope method to exercise training (n = 25) or control group (n = 25). The exercise group underwent an exercise program consisting of 3 visits/week for 3 months. Paired sample t tests were used to look for in-group differences and independent sample t tests for between-group differences. RESULTS: Three months after CRT there were significant improvements in all functional, exercise hemodynamic, and echocardiographic measures. After randomization the exercise group showed further significant improvements in functional, exercise hemodynamic, and QOL measures compared with the control group. There were also significant in-group improvements in peak skeletal muscle function and ejection fraction that did not reach statistical significance on intergroup analysis. CONCLUSIONS: Exercise training leads to further improvements in exercise capacity, hemodynamic measures, and QOL in addition to the improvements seen after CRT. Therefore, exercise training allows maximal benefit to be attained after CRT.

Reduced cardiac functional reserve and quality of life in adults with GH deficiency.

INTRODUCTION: Patients with severe GH deficiency (GHD) suffer with a reduced quality of life in addition to diverse changes in cardiac size and performance. So far, the cardiac reserve ability to maintain the circulation during peak exercise has not been measured. We tested the hypothesis that patients with severe GHD have reduced cardiac reserve function compared with healthy controls and that this could explain, in part, their reduced quality of life. AIMS: Eighteen patients with severe GHD and an assessment of GHD in adults (AGHDA) score > or =11 (mean 20.0, range 12-25) were studied and compared with 18 age-, sex- and body mass index-matched healthy controls. Peak cardiac power and cardiorespiratory fitness were investigated using noninvasive haemodynamic measurements during maximal cardiopulmonary exercise testing. RESULTS: Compared with matched controls, the cardiac power of GHD patients during exercise to volitional exhaustion was significantly reduced by 15% (mean +/- SD 4.4 +/- 1.0 W vs. 5.2 +/- 1.0 W, P = 0.02). Patients with GHD also had lower cardiac chronotropic reserve (peak heart rate 154 +/- 21/min vs. 174 +/- 11/min, P = 0.001) and a lower cardiac pressure-generating capacity (systolic blood pressure 160 +/- 25 mmHg vs. 200 +/- 15 mmHg, P < 0.0001). We found no correlation between any measure of peak cardiac power or function and the AGHDA score. CONCLUSION: Using this robust noninvasive method of assessing functional cardiac pumping capacity, we have for the first time shown that, while patients with severe GHD have a significantly impaired cardiac functional reserve associated with chronotropic incompetence and impaired pressure-generating capacity, this does not correlate with their reduced quality of life assessed using the current standard AGHDA score.

Cardiac functional reserve is diminished in growth hormone-deficient adults.

Various studies have shown that patients with severe growth hormone deficiency (GHD) have diverse changes in left ventricular (LV) size or performance but so far there is no direct indication of cardiac reserve ability to maintain the circulation during peak exercise. We tested the hypothesis that patients with severe GHD have reduced cardiac reserve function compared with healthy controls. Eighteen patients with severe GHD were studied and compared with 18 age-, sex-, and body mass index (BMI)-matched healthy controls. Peak cardiac power and cardiorespiratory fitness were investigated using noninvasive hemodynamic measurements during maximal cardiopulmonary exercise testing. Compared with matched controls, the cardiac power of GHD patients during exercise to volitional exhaustion was significantly reduced by 15% (mean +/- SD: 4.4 +/- 1.0 watts (W) vs. 5.2 +/- 1.0 W, P= 0.02), despite attaining similar aerobic exercise peaks (VO(2max), GHD: 2390 +/- 822 mL/min vs. controls: 2461 +/- 872 mL/min, P= 0.80) and similar peak respiratory exchange ratios. The lower peak cardiac power could not be accounted for by peripheral alterations because both groups reached similar peak exercise systemic vascular resistances. Patients with GHD also had lower cardiac chronotropic reserve (peak heart rate: 154 +/- 21 bpm vs. 174 +/- 11 bpm, P= 0.001) and a lower cardiac pressure-generating capacity (systolic blood pressure [SBP] 160 +/- 25 mmHg vs. 200 +/- 15 mmHg, P < 0.0001). Using this robust noninvasive method of assessing functional cardiac pumping capacity we have for the first time shown that patients with severe GHD have a significantly impaired cardiac functional reserve associated with chronotropic incompetence and impaired pressure-generating capacity.

A study of presbycardia, with gender differences favoring ageing women.

BACKGROUND: The impact of ageing on the human cardiovascular system has been the subject of several studies in recent years, but with insufficient emphasis on defining sex-specific differences. To rectify this, gender-specific differences in structure and function in the human cardiovascular system were studied in a European population during natural ageing. METHODS: Cardiac power output (CPO) was measured and integrated with changes in left ventricular (LV) mass, diastolic, systolic and limb blood flow, blood pressure and exercise capacity in 93 health-screened men and 122 women, aged 20 to 75 years. RESULTS: Correlating with a 21% loss of LV mass, maximum cardiac pumping (i.e. CPOmax=QmaxxMAPmax) and reserve (CR=CPOmax-CPOrest) capacities decreased 20-25% with age in male hearts. In contrast, CPOmax, CR and LV mass were all preserved in ageing women. Maximum cardiac output (Qmax; 26-32%), peak forearm blood flow (FBFpeak; 61%) and exercise capacity (40-50%) all decreased, but more so in men than women. In contrast, systemic vascular resistance (68-75%) and mean arterial pressure (MAPmax; 14-26%) increased in both sexes. CPOrest decreased 27% in men, but was unchanged in women, despite lower early:late diastolic filling (48-51%), Qrest (19-23%) and FBFrest (56%) in both sexes. CONCLUSIONS: Understanding sex-specific differences in cardiovascular ageing is important for public health and biomedical research, given increasingly larger older populations and the need to prevent and treat cardiovascular disease.

Cardiac stem cell-based myocardial regeneration: towards a translational approach.

The adult mammalian heart, including humans, harbors bone fide cardiac stem cells (CSCs) distributed throughout the atria and ventricles. Their discovery almost four years ago initiated a brand new field of cardiac regenerative biology that has profoundly changed the outlook of developmental and adult cardiac biology/physiology and the potential for treating cardiac failure. Indeed, despite its initial hesitance, the research community has now accepted that the heart has an endogenous myocardial regenerative potential owed to CSCs, which challenges the previous accepted notion of the adult heart as a post-mitotic organ. Also, burgeoning evidence is converging to the possibility that CSCs are actually the founder of the heart in the embryonic life. CSCs are involved in cardiac cellular homeostasis during aging and adaptation to physiological and pathological stress. When transplanted into damaged hearts, CSCs have the capacity to generate significant new myocardial tissue and ameliorate ventricular function. However, more relevant for translational research and its application for future regeneration protocols, this reservoir of CSCs can also be activated by local injections of several growth factors or through the administration of systemic drugs, such as statins, to obtain beneficial results similar to those of CSC transplantation. The present review highlights current knowledge on the biology of CSCs and the prospects of CSC activation in situ, without the need for cell expansion in vitro and consequent transplantation.

Acute beta-adrenergic overload produces myocyte damage through calcium leakage from the ryanodine receptor 2 but spares cardiac stem cells.

A hyperadrenergic state is a seminal aspect of chronic heart failure. Also, "Takotsubo stress cardiomyopathy," is associated with increased plasma catecholamine levels. The mechanisms of myocyte damage secondary to excess catecholamine exposure as well as the consequence of this neurohumoral burst on cardiac stem cells (CSCs) are unknown. Cardiomyocytes and CSCs were exposed to high doses of isoproterenol (ISO), in vivo and in vitro. Male Wistar rats received a single injection of ISO (5 mg kg-1) and were sacrificed 1, 3, and 6 days later. In comparison with controls, LV function was impaired in rats 1 day after ISO and started to improve at 3 days. The fraction of dead myocytes peaked 1 day after ISO and decreased thereafter. ISO administration resulted in significant ryanodine receptor 2 (RyR2) hyperphosphorylation and RyR2-calstabin dissociation. JTV519, a RyR2 stabilizer, prevented the ISO-induced death of adult myocytes in vitro. In contrast, CSCs were resistant to the acute neurohumoral overload. Indeed, CSCs expressed a decreased and inverted complement of beta1/beta2-adrenoreceptors and absence of RyR2, which may explain their survival to ISO insult. Thus, a single injection of ISO causes diffuse myocyte death through Ca2+ leakage secondary to the acutely dysfunctional RyR2. CSCs are resistant to the noxious effects of an acute hyperadrenergic state and through their activation participate in the response to the ISO-induced myocardial injury. The latter could contribute to the ability of the myocardium to rapidly recover from acute hyperadrenergic damage.

Anabolic effects of a non-myotoxic dose of the beta2-adrenergic receptor agonist clenbuterol on rat plantaris muscle.

Previous investigations of the effects of clenbuterol have used suprapharmacological doses that induce myocyte death, alter muscle phenotype, and do not approximate the proposed therapeutic dose for humans. Recently, we reported that smaller doses of clenbuterol induce muscle growth without causing myocyte death. In the present study we used histochemical and proteomic techniques to investigate the molecular effects of this dose. Male Wistar rats (n = 6, per group) were infused with saline or 10 microg/kg/day clenbuterol via subcutaneously implanted osmotic pumps. After 14 days the animals' plantaris muscles were isolated for histochemical and proteomic analyses. Clenbuterol induced significant muscle growth with concomitant protein accretion and preferential hypertrophy of fast oxidative glycolytic fibers. Clenbuterol reduced the optical density of mitochondrial staining in fast fibers by 20% and the glycogen content of the muscle by 30%. Differential analysis of two-dimensional gels showed that heat shock protein 72 and beta-enolase increased, whereas aldolase A, phosphogylcerate mutase, and adenylate kinase decreased. Only heat shock protein 72 has previously been investigated in clenbuterol-treated muscles. The clenbuterol-induced increase in muscle growth was concomitant with qualitative changes in the muscle's proteome that need to be considered when proposing therapeutic uses for this agent.

Relative myotoxic and haemodynamic effects of the beta-agonists fenoterol and clenbuterol measured in conscious unrestrained rats.

The beta(2)-adrenoceptor (beta(2)-AR) agonists clenbuterol and fenoterol have similar beneficial effects in animal models of heart failure. However, large doses of clenbuterol can induce cardiomyocyte death, and it is not known which of these agents has the most favourable therapeutic profile. We have investigated the cardiotoxicity of clenbuterol and fenoterol alongside that of isoprenaline, and compared their haemodynamic effects. Wistar rats (n = 6 per group) were subcutaneously injected with each beta-agonist (0.003-3 mmol kg(-1)) or saline, and cardiomyocyte apoptosis was detected by caspase 3 immunohistochemistry. In a separate experiment, rats (n = 4) were given equivalent doses to those used in the myotoxicity studies, in a randomized cross-over design, and their blood pressure recorded via radiotelemetry. Injection of 0.3 mmol kg(-1) fenoterol or isoprenaline, but not clenbuterol, induced significant cardiomyocyte apoptosis (0.4 +/- 0.05%; P < 0.05). At 3 mmol kg(-1), all agonists induced apoptosis (fenoterol, 1.1 +/- 0.1%; isoprenaline, 0.9 +/- 0.8%; and clenbuterol, 0.4 +/- 0.07%; P < 0.05). beta(1)-Adrenoceptor antagonism (10 mg kg(-1) bisoprolol) prevented 92% (P < 0.05) of apoptosis induced by all three agonists, but clenbuterol-induced apoptosis could also be prevented by 96% (P < 0.05) by beta(2)-AR antagonism (10 mg kg(-1) ICI 118 551). Clenbuterol decreased diastolic (1.3- to 1.6-fold; P < 0.05) and systolic blood pressure (1.3-fold; P < 0.05), and doses > 0.3 mmol kg(-1) increased heart rate (1.4-fold; P < 0.05). Fenoterol increased heart rate (1.2- to 1.4-fold; P < 0.05), and doses > 0.3 mmol kg(-1) decreased diastolic blood pressure (1.3-fold; P < 0.05). In conclusion, the cardiotoxicity of fenoterol was similar to isoprenaline and greater than clenbuterol, and fenoterol had less desirable haemodynamic effects.

Dose-dependent separation of the hypertrophic and myotoxic effects of the beta(2)-adrenergic receptor agonist clenbuterol in rat striated muscles.

Muscle growth in response to large doses (milligrams per kilogram) of beta(2)-adrenergic receptor agonists has been reported consistently. However, such doses may also induce myocyte death in the heart and skeletal muscles and hence may not be safe doses for humans. We report the hypertrophic and myotoxic effects of different doses of clenbuterol. Rats were infused with clenbuterol (range, 1 microg to 1 mg.kg(-1)) for 14 days. Muscle protein content, myofiber cross-sectional area, and myocyte death were then investigated. Infusions of >or=10 microg.kg(-1).d(-1) of clenbuterol significantly (P<0.05) increased the protein content of the heart (12%-15%), soleus (12%), plantaris (18%-29%), and tibialis anterior (11%-22%) muscles, with concomitant myofiber hypertrophy. Larger doses (100 microg or 1 mg) induced significant (P<0.05) myocyte death in the soleus (peak 0.2+/-0.1% apoptosis), diaphragm (peak 0.15+/-0.1% apoptosis), and plantaris (peak 0.3+/-0.05% necrosis), and significantly increased the area fraction of collagen in the myocardium. These data show that the low dose of 10 microg.kg(-1).d(-1) can be used in rats to investigate the anabolic effects of clenbuterol in the absence of myocyte death.

Ageing and activity: their effects on the functional reserve capacities of the heart and vascular smooth and skeletal muscles.

During perinatal life striated muscles grow through the acquisition of more contractile cells (myocytes or fibres) followed by their postnatal enlargement (i.e. hypertrophy). In the ageing adult these events are reversed, with a progressive loss of myocytes that cannot be fully compensated despite the presence of cell renewal systems or reactive myocyte hypertrophy. Hence the functional reserve capacities of the heart and skeletal muscles decline with age. This is probably a consequence of physiological ageing and diminished levels of physical activity. As a result daily tasks once taken for granted become progressively more difficult, and eventually impossible, to perform. For example, sufficient coordinated absolute muscle force is required for an individual to rise from a chair or climb stairs, and the reserve capacity of the heart is a major determinant of an individual's ability to remain active and cope with daily stresses and illnesses. Long-term participation in endurance-based activities helps to preserve cardiac reserve, and has both direct and indirect beneficial effects on vascular smooth muscle and health preservation within the cardiovascular system. In contrast, this type of activity does little to protect skeletal muscles against the age-related losses of fast-twitch fibres, small motor units, overall muscle mass and power output. While resistance exercise promotes fibre hypertrophy in skeletal muscles, and to a lesser extent in myocytes of the heart, the explosive power of muscles still declines with age. Hence, while physical activity is important in attenuating age-related changes in muscle function and its reserve capacity, it delays rather than prevents the deleterious effects of ageing per se. Despite this, in a culture where inactivity has become an accepted part of life we still need to explore in greater detail the benefits of habitual physical activity, and use this information as a community-based educational tool to help prevent or delay cardiovascular disease, obesity, arthritis and the frailty associated with old age.

Relative toxicity of cardiotonic agents: some induce more cardiac and skeletal myocyte apoptosis and necrosis in vivo than others.

We sought to determine the relative myotoxicity of a sample of cardiotonic (catecholaminergic and PDE Inhibitory) agents currently available for clinical use. Male Wistar rats (292 +/- 24 g) were administered single subcutaneous injections of 20 mmol kg(-1) of each agent. Myocyte apoptosis (caspase-3 and annexin-V) and necrosis (anti-myosin antibody) were detected immunohistochemically on cryosections of the heart and soleus muscle. All of the cardiotonic agents except dopamine produced significant amounts of cardiomyocyte death compared with the vehicle controls, with necrosis (range 2-8%, p < 0.01) approximately one order of magnitude greater in extent than apoptosis (range 0.06-0.5%, p < 0.05). The incidence of necrosis induced by norepinephrine (8%) was approximately twice that of epinephrine and isoproterenol (4 %) and four times that of dobutamine and milrinone (2%). All agents were also toxic to the soleus muscle (range 0.1-8%), but isoproterenol (8%, p < 0.05) and epinephrine (4%, p < 0.05) were the most significant. No cell death was detected in control animals treated with only the vehicle. A majority of cardiotonic agents currently in clinical use are toxic to cardiac and skeletal myocytes. These observations suggest that judicious clinical use of such agents requires careful weighing of potential benefits against the harm via accelerated cumulative loss of myocytes.

Dose-dependent apoptotic and necrotic myocyte death induced by the beta2-adrenergic receptor agonist, clenbuterol.

We have investigated the dose- and time-dependency of myocyte apoptosis and necrosis induced by the beta2-adrenergic receptor agonist, clenbuterol, with the aim of determining whether myocyte apoptosis and necrosis are two separate processes or a continuum of events. Male Wistar rats were administered subcutaneous injections of clenbuterol, and immunohistochemistry was used to detect myocyte-specific apoptosis and necrosis. Myocyte apoptosis peaked 4 h after, and necrosis 12 h after, clenbuterol administration. In the soleus, peak apoptosis (5.8 +/- 2.0%; P < 0.05) was induced by 10 mug and peak necrosis (7.4 +/- 1.7%; P < 0.05) by 5 mg x kg(-1) clenbuterol. Twelve hours after clenbuterol administration, 73% of damaged myocytes labeled as necrotic, 27% as apoptotic and necrotic, and 0% as purely apoptotic. Administrations of clenbuterol (10 microg x kg(-1)) at 48-h intervals induced cumulative myocyte death over 8 days. These data show that the phenotype of myocyte death is dependent on the magnitude of the insult and the time at which it is investigated. Only very low doses induced apoptosis alone; in most cases apoptotic myocytes lysed and became necrotic and the magnitude of necrosis was greater than that of apoptosis. Thus, it is important to investigate both apoptotic and necrotic myocyte death, contrary to the current trend of only investigating apoptotic cell death.

Angiotensin II induces apoptosis in vivo in skeletal, as well as cardiac, muscle of the rat.

Our previous work has established that angiotensin II is cardiotoxic. Here we sought to investigate whether skeletal muscle is similarly susceptible to damage. Male Wistar rats were either given a single subcutaneous injection of angiotensin II (range 1 microg kg-1 to 10 mg kg-1) or only the vehicle and killed 7 h later, or implanted with preconditioned osmotic pumps dispensing 1 mg kg-1 day-1 angiotensin II and killed 9 or 18 h later. Apoptotic (caspase 3 positive) myocytes were counted on cryosections of the heart, soleus, tibialis anterior and diaphragm muscle. Single injections of 100 microg kg-1 to 10 mg kg-1 angiotensin II induced significant (P<0.05) myocyte apoptosis (per 10(4) viable myocytes) in the heart and this was heterogeneously distributed, peaking (5.7+/-0.6; P<0.05) at a point 6 mm from the apex, i.e. approximately three-quarters of the way towards the base. The slow-twitch soleus muscle was also damaged significantly (peak=2.6+/-0.4; P<0.05), while only the administration of 1 mg kg-1 induced significant (P<0.05) apoptosis in the fast-twitch tibialis anterior muscle (peak=1.2+/-0.3). Infusion of 1 mg kg-1 day-1 angiotensin II induced more myocyte apoptosis than a single bolus administration of the same dose, and in general there was a higher incidence of apoptosis in muscles harvested after 18 than after 9 h. Infusion of 1 mg kg-1 day-1 angiotensin II over 18 h induced significant (P<0.05) myocyte apoptosis in the heart (3.3+/-0.4), soleus (3.9+/-1), tibialis anterior (5.9+/-0.4) and diaphragm (19.8+/-5.6) muscle. Depending on the muscle type, angiotensin II induces myocyte apoptosis in skeletal muscle to a similar or greater extent as in cardiac muscle, supporting the hypothesis that angiotensin II is generally toxic to all striated muscles.

Aldosterone induces myocyte apoptosis in the heart and skeletal muscles of rats in vivo.

Over activation of the renin-angiotensin-aldosterone system is known to be cardiotoxic but the potential injurious effects on the skeletal musculature have not been investigated. Male Wistar rats were given subcutaneous injections of aldosterone (1 microg-10 mg kg-1) and killed 7 h later, or continuous infusion (1 mg kg-1 d-1) and killed 48 h later. The role of the mineralocorticoid receptor in mediating aldosterone-induced apoptosis in vivo was investigated using spironolactone (200 mg kg-1). The number of apoptotic (caspase 3 positive) myocytes was counted on cryosections of the heart, soleus and Tibialis Anterior muscles. Injections of aldosterone induced significant (P<0.05) cardiomyocyte apoptosis (peak=2.46+/-0.6 per 10(4) viable myocytes) over the range of 100 microg-10 mg kg-1, whereas only administration of 1 mg kg-1 induced significant (P<0.05) apoptosis (2.47+/-0.8 per 10(4) viable myocytes) in the soleus muscle. In contrast, no apoptosis was detected in the striated muscles after administration of only the vehicle. Infusion of aldosterone induced less apoptosis than the same dose (1 mg kg-1) given as a single injection. Prior administration of spironolactone significantly (P<0.05) protected the heart (90%) and soleus muscle (79%) against the apoptosis induced by a single injection of 1 mg kg-1 aldosterone. These data confirm a myotoxic effect of aldosterone on the heart and provide the first description of aldosterone-induced myocyte apoptosis in skeletal muscle. High circulating levels of aldosterone are clearly capable of damaging all types of striated muscle and this may lend support to the concept that heart failure is a generalised, rather than cardiac-specific, myopathy.

beta2-Adrenergic receptor stimulation in vivo induces apoptosis in the rat heart and soleus muscle.

High doses of the beta2-adrenergic receptor (AR) agonist clenbuterol can induce necrotic myocyte death in the heart and slow-twitch skeletal muscle of the rat. However, it is not known whether this agent can also induce myocyte apoptosis and whether this would occur at a lower dose than previously reported for myocyte necrosis. Male Wistar rats were given single subcutaneous injections of clenbuterol. Immunohistochemistry was used to detect myocyte-specific apoptosis (detected on cryosections via a caspase 3 antibody and confirmed with annexin V, single-strand DNA labeling, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). Myocyte apoptosis was first detected at 2 h and peaked 4 h after clenbuterol administration. The lowest dose of clenbuterol to induce cardiomyocyte apoptosis was 1 microg/kg, with peak apoptosis (0.35 +/- 0.05%; P < 0.05) occurring in response to 5 mg/kg. In the soleus, peak apoptosis (5.8 +/- 2%; P < 0.05) was induced by the lower dose of 10 microg/kg. Cardiomyocyte apoptosis was detected throughout the ventricles, atria, and papillary muscles. However, this damage was most abundant in the left ventricular subendocardium at a point 1.6 mm, that is, approximately one-quarter of the way, from the apex toward the base. beta-AR antagonism (involving propranolol, bisoprolol, or ICI 118551) or reserpine was used to show that clenbuterol-induced myocardial apoptosis was mediated through neuromodulation of the sympathetic system and the cardiomyocyte beta1-AR, whereas in the soleus direct stimulation of the myocyte beta2-AR was involved. These data show that, when administered in vivo, beta2-AR stimulation by clenbuterol is detrimental to cardiac and skeletal muscles even at low doses, by inducing apoptosis through beta1- and beta2-AR, respectively.

Catecholamine-induced apoptosis and necrosis in cardiac and skeletal myocytes of the rat in vivo: the same or separate death pathways?

High levels of catecholamines are myotoxic but the relative amounts of apoptosis and necrosis have not been established in vivo in cardiac and skeletal muscles. Immunohistochemistry was used to detect and quantify myocyte-specific necrosis (myosin antibody in vivo) and apoptosis (caspase-3 antibody in vitro) in the heart and soleus muscles of male Wistar rats that had received single subcutaneous injections of isoprenaline over the range 1 microg to 5 mg [kg body weight (BW)](-1). Peak myocyte apoptosis occurred 3-6 h after, and necrosis 18 h after, a single injection of 5 mg (kg BW)(-1) isoprenaline in vivo. In the heart myocyte death was mediated through the beta1-adrenergic receptor whereas myocyte death in the soleus muscle was mediated through the beta2-adrenergic receptor. Cardiomyocyte death was heterogeneously distributed throughout the heart, being greatest in the left ventricle (LV) subendocardium and peaking close to the apex, but with significantly more necrosis than apoptosis. Extensive co-localization of caspase-3 and myosin labelling was found in the myocytes of both the heart and the slow-twitch soleus muscle. This, together with similar spatial distributions and responses to catecholamine doses, suggests that either caspase-3 activation occurs in necrotic as well as apoptotic myocytes or that a large proportion of apoptotic myocytes progress to secondary necrosis in vivo.