Testosterone modulates cardiomyocyte Ca(2+) handling and contractile function

The extent to which sex differences in cardiac function may be attributed to the direct myocardial influence of testosterone is unclear. In this study the effects of gonadal testosterone withdrawal (GDX) and replacement (GDX+T) in rats, on cardiomyocyte shortening and intracellular Ca(2+) handling was investigated (0.5 Hz, 25 oC). At all extracellular [Ca(2+)] tested (0.5-2.0 mM), the Ca(2+) transient amplitude was significantly reduced (by approximately 50 %) in myocytes of GDX rats two weeks post-gonadectomy. The time course of Ca(2+) transient decay was significantly prolonged in GDX myocytes (tau, 455+/-80 ms) compared with intact (279+/-23 ms) and GDX+T (277+/-19 ms). Maximum shortening of GDX myocytes was markedly reduced (by more than 60 %) and relaxation significantly delayed (by more than 35 %) compared with intact and GDX+T groups. Thus testosterone replacement completely reversed the cardiomyocyte hypocontractility induced by gonadectomy. These results provide direct evidence for a role of testosterone in regulating functional Ca(2+) handling and contractility in the heart.     

Influence of long-term caloric restriction on myocardial and cardiomyocyte contractile function and autophagy in mice

Both clinical and experimental evidence has revealed that calorie restriction (CR) is capable of improving heart function. However, most the reports are focused on the effect of CR on the pathological states such as obesity, while the effect of CR on heart function in otherwise healthy subjects is not well understood. This study examined the long-term CR effect on cardiac contractile function and possible underlying mechanisms involved. C57BL/6 mice were subjected to a 40% CR or ad libitum feeding for 20 weeks. Echocardiographic and cardiomyocyte contractile properties were evaluated. Intracellular signaling pathways were examined using Western blot analysis. Our results showed that CR overtly lessened glucose intolerance, lessened body and heart weights (although not heart size), lowered fat tissue density, decreased left ventricular (LV) wall thickness (septum and posterior wall) in both systole and diastole, and reduced LV mass (not normalized LV mass) without affecting fractional shortening. Cardiomyocyte cell length and cross-sectional area were reduced, while peak shortening amplitude was increased following CR. CR failed to affect maximal velocity of shortening/relengthening and duration of shortening and relengthening. Immunoblotting data depicted decreased and increased phosphorylation of Akt/glycogen synthase kinase-3β and AMP-dependent protein kinase/acetyl-CoA carboxylase, respectively, following CR. CR also dampened the phosphorylation of mammalian target of rapamycin, extracellular-signal-regulated protein kinase 1/2 and c-Jun, while it increased the phosphorylation of c-Jun NH2-terminal kinase. Last but not least, CR significantly promoted cardiac autophagy as evidenced by increased expression of LC3B-II (and LC3B-II to LC3B-I ratio) and Beclin-1. In summary, our data suggested that long-term CR may preserve cardiac contractile function with improved cardiomyocyte function, lessen cardiac remodeling and promote autophagy.     

Impaired sarcoplasmic reticulum function leads to contractile dysfunction and cardiac hypertrophy

Sarcoplasmic reticulum (SR)-mediated Ca(2+) sequestration and release are important determinants of cardiac contractility. In end-stage heart failure SR dysfunction has been proposed to contribute to the impaired cardiac performance. In this study we tested the hypothesis that a targeted interference with SR function can be a primary cause of contractile impairment that in turn might alter cardiac gene expression and induce cardiac hypertrophy. To study this we developed a novel animal model in which ryanodine, a substance that alters SR Ca(2+) release, was added to the drinking water of mice. After 1 wk of treatment, in vivo hemodynamic measurements showed a 28% reduction in the maximum speed of contraction (+dP/dt(max)) and a 24% reduction in the maximum speed of relaxation (-dP/dt(max)). The slowing of cardiac relaxation was confirmed in isolated papillary muscles. The late phase of relaxation expressed as the time from 50% to 90% relaxation was prolonged by 22%. After 4 wk of ryanodine administration the animals had developed a significant cardiac hypertrophy that was most prominent in both atria (right atrium +115%, left atrium +100%, right ventricle +23%, and left ventricle +13%). This was accompanied by molecular changes including a threefold increase in atrial natriuretic factor mRNA and a sixfold increase in beta-myosin heavy chain mRNA. Sarcoplasmic endoplasmic reticulum Ca(2+) mRNA was reduced by 18%. These data suggest that selective impairment of SR function in vivo can induce changes in cardiac gene expression and promote cardiac growth.     

Adenosine kinase regulation of cardiomyocyte hypertrophy

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKT(Ser??3) phosphorylation but did attenuate sustained phosphorylation of Raf(Ser33?) (24-48 h), mTOR(Ser2???) (24-48 h), p70S6k(Thr3??) (2.5-48 h), and ERK(Thr2?2/Tyr2??) (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k(Thr3??) phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK(Thr202/Tyr204) and AKT(Ser??3). Reduction of Raf-induced p70S6k(Thr3??) phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of Raf signaling to mTOR/p70S6k.     

The effects of sarpogrelate on cardiomyocyte hypertrophy

Sarpogrelate was developed as an antiplatelet agent antagonizing 5-hydroxytryptamine (5-HT) receptors. It had been reported that 5-HT receptors were expressed in cardiovascular system, and that sarpogrelate had antihypertrophic effects in vascular smooth muscle cells. Cardiac hypertrophy is a major problem in cardiac diseases, so the present study was designed to elucidate the effects of sarpogrelate on cardiac hypertrophy. Cultured rat cardiomyocytes (MCs) and cardiac nonmyocytes (NMCs) were prepared by Percoll gradient and adhesion method and MCs were incubated with (MCs/NMCs) or without NMCs. As an index of protein synthesis of MCs, [3H]-leucine uptake into MCs and MCs/NMCs was measured. Sarpogrelate decreased [3H]-leucine uptake into MCs (maximum 62.6+/-20.6% of control at 10(-4)M, p<0.05 vs. control). Sarpogrelate also significantly attenuated angiotensin-II- and endothelin-1-induced [3H]-leucine uptake. These results indicated that sarpogrelate might have antihypertrophic effects and could be a useful aid for cardiovascular disease.     

Propofol depresses angiotensin II-induced cardiomyocyte hypertrophy in vitro

Cardiomyocyte hypertrophy is formed in response to pressure or volume overload, injury, or neurohormonal activation. The most important vascular hormone that contributes to the development of hypertrophy is angiotensin II (Ang II). Accumulating studies have suggested that reactive oxygen species (ROS) may play an important role in cardiac hypertrophy. Propofol is a general anesthetic that possesses antioxidant action. We therefore examined whether propofol inhibited Ang II-induced cardiomyocyte hypertrophy. Our results showed that both ROS formation and hypertrophic responses induced by Ang II in cardiomyocytes were partially blocked by propofol. Further studies showed that propofol inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2) induced by Ang II via a decrease in ROS production. In addition, propofol also markedly attenuated Ang II-stimulated nuclear factor-kappaB (NF-kappaB) activation via a decrease in ROS production. In conclusion, propofol prevents cardiomyocyte hypertrophy by interfering with the generation of ROS and involves the inhibition of the MEK/ERK signaling transduction pathway and NF-kappaB activation.     

Cardiac overexpression of metallothionein rescues cold exposure-induced myocardial contractile dysfunction through attenuation of cardiac fibrosis despite cardiomyocyte mechanical anomalies

Cold exposure is associated with an increased prevalence of cardiovascular disease although the mechanism is unknown. Metallothionein, a heavy-metal-scavenging antioxidant, protects against cardiac anomalies. This study was designed to examine the impact of metallothionein on cold exposure-induced myocardial dysfunction, intracellular Ca(2+) derangement, fibrosis, endoplasmic reticulum (ER) stress, and apoptosis. Echocardiography, cardiomyocyte function, and Masson trichrome staining were evaluated in Friend virus B (FVB) and cardiac-specific metallothionein transgenic mice after cold exposure (3 months, 4 °C). Cold exposure increased plasma levels of norepinephrine, endothelin-1, and TGF-β; reduced plasma NO levels and cardiac antioxidant capacity; enlarged ventricular end-systolic diameter; compromised fractional shortening; promoted reactive oxygen species (ROS) production and apoptosis; and suppressed the ER stress markers Bip, calregulin, and phospho-eIF2α, accompanied by cardiac fibrosis and elevated levels of matrix metalloproteinases and Smad-2/3 in FVB mice. Cold exposure-induced echocardiographic, histological, ER stress, ROS, apoptotic, and fibrotic signaling changes (but not plasma markers) were greatly improved by metallothionein. In vitro metallothionein induction by zinc chloride ablated H(2)O(2)- but not TGF-β-induced cell proliferation in fibroblasts. In summary, our data suggest that metallothionein protects against cold exposure-induced cardiac anomalies possibly through attenuation of myocardial fibrosis.     

Neuronostatin, a novel peptide encoded by somatostatin gene, regulates cardiac contractile function and cardiomyocyte survival

Neuronostatin, a recently discovered peptide encoded by somatostatin gene, is involved in regulation of neuronal function, blood pressure, food intake, and drinking behavior. However, the biological effects of neuronostatin on cardiac myocytes are not known, and the intracellular signaling mechanisms induced by neuronostatin remain unidentified. We analyzed the effect of neuronostatin in isolated perfused rat hearts and in cultured primary cardiomyocytes. Neuronostatin infusion alone had no effect on left ventricular (LV) contractile function or on isoprenaline- or preload-induced increase in cardiac contractility. However, infusion of neuronostatin significantly decreased the positive inotropic response to endothelin-1 (ET-1). This was associated with an increase in phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK). Treatment of both neonatal and adult cardiomyocytes with neuronostatin resulted in reduced cardiomyocyte viability. Inhibition of JNK further increased the neuronostatin-induced cell death. We conclude that neuronostatin regulates cardiac contractile function and cardiomyocyte survival. Receptors for neuronostatin need to be identified to further characterize the biological functions of the peptide.     

Akt and PI 3-kinase signaling in cardiomyocyte hypertrophy and survival

In many systems, activation of the "protein and lipid kinase" phosphoinositide 3-kinase (PI 3-kinase) and its downstream serine-threonine kinase effector, Akt (or Protein Kinase B), provide a potent stimulus for cell proliferation, growth, and survival. In the heart, constrained by the limited proliferative capacity of cardiomyocytes, this pathway plays a key role in regulating cardiomyocyte growth and survival, with little effect on proliferation. Simultaneously, PI 3-kinase and Akt are important modulators of metabolic substrate utilization and cardiomyocyte function. Thus, the convergent signaling pathways controlling so many clinically important phenotypes of the cardiomyocyte suggest it holds promise as a therapeutic target in a variety of cardiac diseases. However, the similar role of PI 3-kinase/Akt signaling in neoplasia suggests the difficulty of activating this pathway in the heart without invoking adverse consequences elsewhere. Here we review evidence regarding the role of PI 3-kinase/Akt in controlling cardiomyocyte growth and survival, and discuss the implications for therapeutic strategies.     

Cardiac contractile proteins and autoimmune myocarditis

Concerning cardiac contractile proteins, antigenicity and myocarditogenicity were discussed. In normal states, these proteins are immunologically tolerant, and can not provoke any heart-specific disease. Why the proteins can provoke such lethal autoimmune myocarditis has not been completely elucidated. Shortly after cardiac infection or myocardial ischemia, these proteins may work as the antigen for the autoimmune myocardites. First of all, the role of cardiac myosin has been strongly emphasized. But, the antigen determinants: epitope proteins remain unclear. Either cross-activity to the streptococcal M protein and/or the -helical coiled-coil protein may be an important factor to determine antigenicity. In this autoimmune myocarditis, the roles of T-lymphocyte and cardiac dendritic cell are noticeable. Through further study on the relation between antigen epitope and the infectious agents in the heart; on cardio-cytotoxity of the T-lymphocyte and on the precise contribution of cardiac dendritic cells, this autoimmune myocarditis will be more clarified.     

Extracellular matrix dependence of organization of the cardiomyocyte contractile apparatus

In the process of cardiomyocyte cultivation, there occurs rearrangement of their contractile apparatus with transformation of typical myofibrils into structures of nonmuscle type and with subsequent restoration of the initial organization. The causes and mechanisms of the rearrangements described are unknown. In this study, it was shown that, at cultivation of cardiomyocytes on the individual proteins of the extracellular matrix (ECM), as well as on the matrix produced either by cardiac fibroblasts or by similar cardiomyocytes, the period in which the contractile apparatus is in a converted state is shortened, whereas soluble factors of the conditioned cultural medium do not produce any obvious effect on the above-indicated rearrangements. Using the method of extracellular matrix isolation adapted for cardiomyocytes, we have confirmed that, in the process of cultivation, cardiomyocytes themselves produce ECM that differs from ECM of cardiac fibroblasts by composition and varies with culturing time.     

CaMKII activates ASK1 and NF-kappaB to induce cardiomyocyte hypertrophy

Ca2+/calmodulin-dependent protein kinase (CaMK) is an important downstream target of Ca2+ in the hypertrophic signaling pathways. We previously showed that the activation of apoptosis signal-regulating kinase 1 (ASK1) or NF-kappaB is sufficient for cardiomyocyte hypertrophy. Infection of isolated neonatal cardiomyocytes with an adenoviral vector expressing CaMKIIdelta3 (AdCaMKIIdelta3) induced the activation of ASK1, while KN93, an inhibitor of CaMKII, inhibited phenylephrine-induced ASK1 activation. Overexpression of CaMKIIdelta3 induced characteristic features of in vitro cardiomyocyte hypertrophy. Infection of cardiomyocytes with an adenoviral vector expressing a dominant negative mutant of ASK1 (AdASK(KM)) inhibited the CaMKIIdelta3-induced hypertrophic responses. Overexpression of CaMKIIdelta3 increased the kappaB-dependent promoter/luciferase activity and induced IkappaBalpha degradation. Coinfection with AdCaMKIIdelta3 and AdASK(KM), and pre-incubation with KN93 attenuated CaMKIIdelta3- and phenylephrine-induced NF-kappaB activation, respectively. Expression of a degradation resistant mutant of IkappaBalpha inhibited CaMKIIdelta3-induced hypertrophic responses. These results indicate that CaMKIIdelta3 induces cardiomyocyte hypertrophy mediated through ASK1-NF-kappaB signal transduction pathway.     

High-dose benfotiamine rescues cardiomyocyte contractile dysfunction in streptozotocin-induced diabetes mellitus.

Diabetic cardiomyopathy is characterized by cardiac dysfunction. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse cardiomyocytes. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg ip). Fourteen days later, control and diabetic (fasting plasma glucose > 13.9 mM) mice were put on benfotiamine therapy (100 mg.kg(-1).day(-1) ip) for another 14 days. Mechanical and intracellular Ca2+ properties were evaluated in left ventricular myocytes using an IonOptix MyoCam system. The following indexes were evaluated: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening, resting and rise of intracellular Ca2+ in response to electrical stimulus, sarcoplasmic reticulum (SR) Ca2+ load, and intracellular Ca2+ decay rate (tau). Two- or four-week STZ treatment led to hyperglycemia, prolonged TPS and TR90, reduced SR Ca2+ load, elevated resting intracellular Ca2+ level and prolonged tau associated with normal PS, maximal velocity of shortening/relengthening, and intracellular Ca2+ rise in response to electrical stimulus. Benfotiamine treatment abolished prolongation in TPS, TR90, and tau, as well as reduction in SR Ca2+ load without affecting hyperglycemia and elevated resting intracellular Ca2+. Diabetes triggered oxidative stress, measured by GSH-to-GSSG ratio and formation of advanced glycation end product (AGE) in the hearts. Benfotiamine treatment alleviated oxidative stress without affecting AGE or protein carbonyl formation. Collectively, our results indicated that benfotiamine may rescue STZ-induced cardiomyocyte dysfunction but not AGE formation in short-term diabetes.     

Neuropeptide Y induces cardiomyocyte hypertrophy via calcineurin signaling in rats

Neuropeptide Y (NPY) has been shown to participate in cardiac hypertrophy. However, the mechanisms by which NPY induces cardiomyocyte hypertrophy are poorly understood. This study tested the hypothesis that NPY induces cardiomyocyte hypertrophy through Ca2+/CaM-dependent calcineurin (CaN) pathway in cultured neonatal rat cardiomyocytes. After 24-h treatment, NPY (100 nM) significantly increased 3H-leucine incorporation and c-Jun mRNA expression, concomitant with augment of CaN activity and protein level in cardiomyocytes compared to those cells without NPY treatment. The enhancement of 3H-leucine incorporation and c-Jun mRNA expression in cardiomyocytes treated with NPY were markedly inhibited by cyclosporine A (CsA), a selective inhibitor of CaN. We also investigated the effect of NPY on intracellular Ca2+ level in cardiomyocytes. There were no obvious changes in intracellular Ca2+ level of cytoplasm and nucleus in cardiomyocytes treated with NPY (100 nM) for 10 min. However, NPY significantly increased intracellular Ca2+ level of cytoplasm and nucleus in cardiomyocytes after 24-h treatment. The result suggested that NPY could induce hypertrophy of cardiomyocytes via Ca2+/CaM-dependent CaN signal pathway. The enhancement of [Ca2+]i caused by NPY may activate CaN signal pathways to mediate cardiac hypertrophy.