Alteration in mitochondrial Ca<Superscript>2+</Superscript> uptake disrupts insulin signaling in hypertrophic cardiomyocytes

Background

Cardiac hypertrophy is characterized by alterations in both cardiac bioenergetics and insulin sensitivity. Insulin promotes glucose uptake by cardiomyocytes and its use as a substrate for glycolysis and mitochondrial oxidation in order to maintain the high cardiac energy demands. Insulin stimulates Ca²⁺ release from the endoplasmic reticulum, however, how this translates to changes in mitochondrial metabolism in either healthy or hypertrophic cardiomyocytes is not fully understood.

Results

In the present study we investigated insulin-dependent mitochondrial Ca²⁺ signaling in normal and norepinephrine or insulin like growth factor-1-induced hypertrophic cardiomyocytes. Using mitochondrion-selective Ca²⁺-fluorescent probes we showed that insulin increases mitochondrial Ca²⁺ levels. This signal was inhibited by the pharmacological blockade of either the inositol 1,4,5-triphosphate receptor or the mitochondrial Ca²⁺ uniporter, as well as by siRNA-dependent mitochondrial Ca²⁺ uniporter knockdown. Norepinephrine-stimulated cardiomyocytes showed a significant decrease in endoplasmic reticulum-mitochondrial contacts compared to either control or insulin like growth factor-1-stimulated cells. This resulted in a reduction in mitochondrial Ca²⁺ uptake, Akt activation, glucose uptake and oxygen consumption in response to insulin. Blocking mitochondrial Ca²⁺ uptake was sufficient to mimic the effect of norepinephrine-induced cardiomyocyte hypertrophy on insulin signaling.

Conclusions

Mitochondrial Ca²⁺ uptake is a key event in insulin signaling and metabolism in cardiomyocytes.

     

Dynasore Protects Mitochondria and Improves Cardiac Lusitropy in Langendorff Perfused Mouse Heart

Background

Heart failure due to diastolic dysfunction exacts a major economic, morbidity and mortality burden in the United States. Therapeutic agents to improve diastolic dysfunction are limited. It was recently found that Dynamin related protein 1 (Drp1) mediates mitochondrial fission during ischemia/reperfusion (I/R) injury, whereas inhibition of Drp1 decreases myocardial infarct size. We hypothesized that Dynasore, a small noncompetitive dynamin GTPase inhibitor, could have beneficial effects on cardiac physiology during I/R injury.

Methods and Results

In Langendorff perfused mouse hearts subjected to I/R (30 minutes of global ischemia followed by 1 hour of reperfusion), pretreatment with 1 µM Dynasore prevented I/R induced elevation of left ventricular end diastolic pressure (LVEDP), indicating a significant and specific lusitropic effect. Dynasore also decreased cardiac troponin I efflux during reperfusion and reduced infarct size. In cultured adult mouse cardiomyocytes subjected to oxidative stress, Dynasore increased cardiomyocyte survival and viability identified by trypan blue exclusion assay and reduced cellular Adenosine triphosphate(ATP) depletion. Moreover, in cultured cells, Dynasore pretreatment protected mitochondrial fragmentation induced by oxidative stress.

Conclusion

Dynasore protects cardiac lusitropy and limits cell damage through a mechanism that maintains mitochondrial morphology and intracellular ATP in stressed cells. Mitochondrial protection through an agent such as Dynasore can have clinical benefit by positively influencing the energetics of diastolic dysfunction.     

Soluble Adenylyl Cyclase Controls Mitochondria-dependent Apoptosis in Coronary Endothelial Cells

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.Increasing evidence suggests that apoptosis of endothelial cells (EC)³ may be responsible for acute and chronic vascular diseases, e.g. through atherogenesis (1), endothelial dysfunction (2), or thrombosis (3). Within several signaling mechanisms, a cAMP-dependent signaling pathway plays a substantial role in mediating apoptotic cell death induced by various stress factors. Elevation of the cellular cAMP either by forskolin-induced stimulation of the G-protein-responsive transmembrane adenylyl cyclase (tmAC) or by treatment with cAMP analogs has been shown to lead to both induction and suppression of apoptosis in different cell types (4–7). This discrepancy may be due to differences in cell types and experimental models. Alternatively, a lack of specificity of tmAC-induced signals, especially directed to distant intracellular targets like mitochondria, may be a cause of the discrepancy. Indeed, the classical model of cAMP signaling requires the diffusion of cAMP from plasma membrane-localized tmAC to targets localized throughout the cell. Diffusion of cAMP throughout the cytosol makes it difficult to selectively activate distally localized targets without also activating more proximal targets. Therefore, such diffusion of cAMP would likely diminish specificity, selectivity, and signal strength. This model is further complicated by the presence of phosphodiesterases, which degrade cAMP, thus preventing its diffusion.In addition to tmAC, a second source of cAMP, soluble adenylyl cyclase (sAC), was demonstrated for mammalian cells (8, 9). Cytosolic localization of sAC provides both specificity and selectivity by permitting generation of cAMP proximal to intracellular targets. Furthermore, this model for cAMP action incorporates phosphodiesterases, which would act to limit diffusion and prevent nonspecific effector activation.Whether sAC participates in apoptosis was unknown. A previous report demonstrated that sAC is co-localized with mitochondria (10). Because mitochondria play a fundamental role in apoptosis (11), we hypothesized that sAC may influence the development of apoptosis by modulating the mitochondrial pathway of apoptosis. Therefore, we aimed to examine the role of sAC in apoptotic cell death, especially its role in the modulation of the mitochondria-dependent pathway of apoptosis. For this purpose, apoptosis was induced in rat coronary EC by simulated in vitro ischemia or by acidosis. By applying pharmacological inhibition of sAC or small interfering RNA (siRNA)-mediated sAC knockdown, we found that sAC activity is required for the induction of apoptosis by ischemia or acidosis. Additionally, translocation of sAC to mitochondria and the sAC-dependent release of cytochrome c suggest that this cyclase specifically regulates the mitochondrial pathway of apoptosis.     

Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes

Background

Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D₂ receptors are expressed in cardiac tissues. However, the roles of dopamine D₂ receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D₂ receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.

Methods

Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.

Results

Treatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca²⁺]_i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.

Conclusions

These data suggest that activation of dopamine D₂ receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.     

The Inotropic Effect of the Active Metabolite of Levosimendan,OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

Aims

We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca²⁺ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.

Methods

Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.

Results

OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca²⁺. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α₁-AR response.

Conclusion

The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca²⁺ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.     

Regulation of Epithelial Na+ Transport by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells

Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Na⁺ transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCD_c14) cells. These cells exhibit aldosterone-stimulated transepithelial Na⁺ currents that depend on both the apical epithelial Na⁺ channel (ENaC) and basolateral Na⁺,K⁺-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (I_sc) in mpkCCD_c14 cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced I_sc in these cells by 50-60% within 30 min. 8-Bromoadenosine-3′,5′-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na⁺ current. Aldosterone doubled ENaC-dependent I_sc over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to I_sc was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na⁺ current was fully inhibited by basolateral ouabain treatment, suggesting that the Na⁺,K⁺-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCD_c14 cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na⁺,K⁺-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Na⁺ reabsorption in the kidney collecting duct, acting to enhance Na⁺,K⁺-ATPase activity.Maintenance of intracellular pH depends in part on the extracellular to intracellular Na⁺ gradient, and elevation of intracellular [Na⁺] can lead to acidification of the cytoplasm. It has been shown that acidification of the cytoplasm of cells from frog skin and toad bladder by increased partial pressure of CO₂ reduces Na⁺ transport and permeability (1, 2). Conversely, the rise in plasma bicarbonate caused by metabolic alkalosis with chronic diuretic use has been shown to increase net renal Na⁺ reabsorption independently of volume status, electrolyte depletion, and/or increased aldosterone secretion (3, 4). However, the underlying mechanisms involved in these phenomena remain unclear.The soluble adenylyl cyclase (sAC)² is a chemosensor that mediates the elevation of cAMP in intracellular microdomains (5-7). Unlike transmembrane adenylyl cyclases (tmACs), sAC is insensitive to regulation by forskolin or heterotrimeric G proteins (8) and is directly activated by elevations of intracellular calcium (9, 10) and/or bicarbonate ions (11). Thus, sAC mediates localized intracellular increases in cAMP in response to variations in bicarbonate levels or its closely related parameters, partial pressure of CO₂ and pH. Mammalian sAC is more similar to bicarbonate-regulated cyanobacterial adenylyl cyclases than to other mammalian nucleotidyl cyclases, which may indicate that there is a unifying mechanism for the regulation of cAMP signaling by bicarbonate across biological systems. Although sAC appears to be encoded by a single gene, there is significant isoform diversity for this ubiquitously expressed enzyme (11, 12) generated by alternative splicing (reviewed in Ref. 13). sAC has been shown to regulate the subcellular localization and/or activity of membrane transport proteins such as the vacuolar H⁺-ATPase (V-ATPase) and cystic fibrosis transmembrane conductance regulator in epithelial cells (14, 15). Functional activity of sAC has been reported in the kidney (16), and sAC has been localized to epithelial cells in the distal nephron (14, 17).Given that natriuresis is decreased during metabolic alkalosis, when bicarbonate is elevated, and Na⁺ reabsorption is impaired by high partial pressure of CO₂, we hypothesized that bicarbonate-regulated sAC may play a key role in the regulation of transepithelial Na⁺ transport in the distal nephron. Reabsorption of Na⁺ in the kidney and other epithelial tissues is mediated by the parallel operation of apical ENaC and basolateral Na⁺,K⁺-ATPase, and both transport proteins can be stimulated by cAMP via the cAMP-dependent protein kinase (PKA) (18, 53). The aims of this study were to investigate the role of sAC in the regulation of transepithelial Na⁺ transport in the kidney through the use of specific sAC inhibitors and electrophysiological measurements. We found that sAC inhibition blocks transepithelial Na⁺ reabsorption in polarized mpkCCD_c14 cells under both basal and hormone-stimulated conditions. Selective membrane permeabilization studies revealed that although ENaC activity appears to be unaffected by sAC inhibition, flux through the Na⁺,K⁺-ATPase is sensitive to sAC modulation. Inhibiting sAC decreases ATPase activity without affecting plasma membrane expression of the pump; thus, tonic sAC activity appears to be required for Na⁺ reabsorption in kidney collecting duct.     

A Low-Dose β1-Blocker in Combination with Milrinone Improves Intracellular Ca2+ Handling in Failing Cardiomyocytes by Inhibition of Milrinone-Induced Diastolic Ca2+ Leakage from the Sarcoplasmic Reticulum

Objectives

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.

Background

The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca²⁺ handling in heart failure remains unclear.

Methods

We investigated the effect of milrinone plus landiolol on intracellular Ca²⁺ transient (CaT), cell shortening (CS), the frequency of diastolic Ca²⁺ sparks (CaSF), and sarcoplasmic reticulum Ca²⁺ concentration ({Ca²⁺}_SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).

Results

In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca²⁺}_SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca²⁺}_SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.

Conclusion

A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca²⁺ leak.     

Reperfusion Promotes Mitochondrial Dysfunction following Focal Cerebral Ischemia in Rats

Background and Purpose

Mitochondrial dysfunction has been implicated in the cell death observed after cerebral ischemia, and several mechanisms for this dysfunction have been proposed. Reperfusion after transient cerebral ischemia may cause continued and even more severe damage to the brain. Many lines of evidence have shown that mitochondria suffer severe damage in response to ischemic injury. The purpose of this study was to observe the features of mitochondrial dysfunction in isolated mitochondria during the reperfusion period following focal cerebral ischemia.

Methods

Male Wistar rats were subjected to focal cerebral ischemia. Mitochondria were isolated using Percoll density gradient centrifugation. The isolated mitochondria were fixed for electron microscopic examination; calcium-induced mitochondrial swelling was quantified using spectrophotometry. Cyclophilin D was detected by Western blotting. Fluorescent probes were used to selectively stain mitochondria to measure their membrane potential and to measure reactive oxidative species production using flow cytometric analysis.

Results

Signs of damage were observed in the mitochondrial morphology after exposure to reperfusion. The mitochondrial swelling induced by Ca²⁺ increased gradually with the increasing calcium concentration, and this tendency was exacerbated as the reperfusion time was extended. Cyclophilin D protein expression peaked after 24 hours of reperfusion. The mitochondrial membrane potential was decreased significantly during the reperfusion period, with the greatest decrease observed after 24 hours of reperfusion. The surge in mitochondrial reactive oxidative species occurred after 2 hours of reperfusion and was maintained at a high level during the reperfusion period.

Conclusions

Reperfusion following focal cerebral ischemia induced significant mitochondrial morphological damage and Ca²⁺-induced mitochondrial swelling. The mechanism of this swelling may be mediated by the upregulation of the Cyclophilin D protein, the destruction of the mitochondrial membrane potential and the generation of excessive reactive oxidative species.     

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and risk of arsenic-induced urothelial carcinoma in residents of southwestern Taiwan

Background

Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D₂ receptors are expressed in cardiac tissues. However, the roles of dopamine D₂ receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D₂ receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.

Methods

Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.

Results

Treatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca²⁺]_i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.

Conclusions

These data suggest that activation of dopamine D₂ receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.     

Cardiac-Specific Overexpression of Metallothionein Rescues against Cigarette Smoking Exposure-Induced Myocardial Contractile and Mitochondrial Damage

Objectives

Second hand cigarette smoke is an independent risk factor for cardiovascular disease. Although a tie between smoking and cardiovascular disease is well established, the underlying mechanisms still remains elusive due to the lack of adequate animal models. This study was designed to use a mouse model of exposure to cigarette smoke, a surrogate of environmental tobacco smoke, to evaluate the impact of cardiac overexpression of heavy metal scavenger metallothionein on myocardial geometry, contractile and intracellular Ca²⁺ properties and apoptosis following side-stream smoke exposure.

Methods

Adult male wild-type FVB and metallothionein transgenic mice were placed in a chamber exposed to cigarette smoke for 1 hour daily for 40 days. Echocardiographic, cardiomyocyte contractile and intracellular Ca²⁺ properties, fibrosis, apoptosis and mitochondrial damage were examined.

Results

Our data revealed that smoke exposure enlarged ventricular end systolic and diastolic diameters, reduced myocardial and cardiomyocyte contractile function, disrupted intracellular Ca²⁺ homeostasis, facilitated fibrosis, apoptosis and mitochondrial damage (cytochrome C release and aconitase activity), the effects of which were attenuated or mitigated by metallothionein. In addition, side-stream smoke expose enhanced phosphorylation of Akt and GSK3β without affecting pan protein expression in the heart, the effect of which was abolished or ameliorated by metallothionein. Cigarette smoke extract interrupted cardiomyocyte contractile function and intracellular Ca²⁺ properties, the effect of which was mitigated by wortmannin and NAC.

Conclusions

These data suggest that side-stream smoke exposure led to myocardial dysfunction, intracellular Ca²⁺ mishandling, apoptosis, fibrosis and mitochondrial damage, indicating the therapeutic potential of antioxidant against in second smoking-induced cardiac defects possibly via mitochondrial damage and apoptosis.     

Somatic 'soluble' adenylyl cyclase isoforms are unaffected in Sacy tm1Lex/Sacy tm1Lex 'knockout' mice

Background

Mammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy) represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacy^tm1Lex/Sacy^tm1Lex knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference.

Principal Findings

We now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which ‘escapes’ the design of the Sacy^tm1Lex knockout allele.

Conclusions/Significance

These studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.     

Inhibition of protein kinase C βII isoform ameliorates methylglyoxal advanced glycation endproduct-induced cardiomyocyte contractile dysfunction

Aims

Accumulation of advanced glycation endproduct (AGE) contributes to diabetic complication including diabetic cardiomyopathy although the precise underlying mechanism still remains elusive. Recent evidence depicted a pivotal role of protein kinase C (PKC) in diabetic complications. To this end, this study was designed to examine if PKCβII contributes to AGE-induced cardiomyocyte contractile and intracellular Ca^2 + aberrations.

Main methods

Adult rat cardiomyocytes were incubated with methylglyoxal-AGE (MG-AGE) in the absence or presence of the PKCβII inhibitor LY333531 for 12 h. Contractile and intracellular Ca^2 + properties were assessed using an IonOptix system including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), rise in intracellular Ca^2 + Fura-2 fluorescence intensity and intracellular Ca^2 + decay. Oxidative stress, O₂⁻ production and mitochondrial integrity were examined using TBARS, fluorescence imaging, aconitase activity and Western blotting.

Key findings

MG-AGE compromised contractile and intracellular Ca^2 + properties including reduced PS, ± dL/dt, prolonged TPS and TR₉₀, decreased electrically stimulated rise in intracellular Ca^2 + and delayed intracellular Ca^2 + clearance, the effects of which were ablated by the PKCβII inhibitor LY333531. Inhibition of PKCβII rescued MG-AGE-induced oxidative stress, O₂⁻ generation, cell death, apoptosis and mitochondrial injury (reduced aconitase activity, UCP-2 and PGC-1α). In vitro studies revealed that PKCβII inhibition-induced beneficial effects were replicated by the NADPH oxidase inhibitor apocynin and were mitigated by the mitochondrial uncoupler FCCP.

Significance

These findings implicated the therapeutic potential of specific inhibition of PKCβII isoform in the management of AGE accumulation-induced myopathic anomalies.     

Stimulation of σ1-receptor restores abnormal mitochondrial Ca mobilization and ATP production following cardiac hypertrophy

Background

We previously reported that the σ₁-receptor (σ₁R) is down-regulated following cardiac hypertrophy and dysfunction in transverse aortic constriction (TAC) mice. Here we address how σ₁R stimulation with the selective σ₁R agonist SA4503 restores hypertrophy-induced cardiac dysfunction through σ₁R localized in the sarcoplasmic reticulum (SR).

Methods

We first confirmed anti-hypertrophic effects of SA4503 (0.1–1 μM) in cultured cardiomyocytes exposed to angiotensin II (Ang II). Then, to confirm the ameliorative effects of σ₁R stimulation in vivo, we administered SA4503 (1.0 mg/kg) and the σ₁R antagonist NE-100 (1.0 mg/kg) orally to TAC mice for 4 weeks (once daily).

Results

σ₁R stimulation with SA4503 significantly inhibited Ang II-induced cardiomyocyte hypertrophy. Ang II exposure for 72 h impaired phenylephrine (PE)-induced Ca^2 + mobilization from the SR into both the cytosol and mitochondria. Treatment of cardiomyocytes with SA4503 largely restored PE-induced Ca^2 + mobilization into mitochondria. Exposure of cardiomyocytes to Ang II for 72 h decreased basal ATP content and PE-induced ATP production concomitant with reduced mitochondrial size, while SA4503 treatment completely restored ATP production and mitochondrial size. Pretreatment with NE-100 or siRNA abolished these effects. Chronic SA4503 administration also significantly attenuated myocardial hypertrophy and restored ATP production in TAC mice. SA4503 administration also decreased hypertrophy-induced impairments in LV contractile function.

Conclusions

σ₁R stimulation with the specific agonist SA4503 ameliorates cardiac hypertrophy and dysfunction by restoring both mitochondrial Ca^2 + mobilization and ATP production via σ₁R stimulation.

General significance

Our observations suggest that σ₁R stimulation represents a new therapeutic strategy to rescue the heart from hypertrophic dysfunction.     

Alpha1 catalytic subunit of AMPK modulates contractile function of cardiomyocytes through phosphorylation of troponin I

Aims

The specific role of AMPKα1 or AMPKα2 in mediating cardiomyocyte contractile function remains elusive. The present study investigated how AMPK activation modulates the contractility of isolated cardiomyocytes.

Main methods

Mechanical properties and intracellular Ca^2 + properties were measured in isolated cardiomyocytes. The stress signaling was evaluated using western blot and immunoprecipitation analysis.

Key findings

AMPK activator, A-769662 induced maximal velocity of shortening (+ dL/dt) and relengthening (− dL/dt), peak height and peak shortening (PS) amplitude in both WT and AMPKα2 KO cardiomyocytes, but did not affect time-to-90% relengthening (TR90). AMPK KD cardiomyocytes demonstrated contractile dysfunction compared with cardiomyocytes from WT and AMPKα2 KO hearts. However, the rise of intracellular Ca^2 + levels as well as intracellular ATP levels has no significant difference among WT, AMPKα2 KO and AMPK KD groups with and without the presence of A-769662. Besides, WT, AMPKα2 KO and AMPK KD group displayed a phosphorylated AMPK and downstream acetyl-CoA carboxylase (ACC) phosphorylation. Interestingly, A-769662 also triggered troponin I (cTnI) phosphorylation at Ser¹⁴⁹ site which is related to contractility of cardiomyocytes. Furthermore, the immunoprecipitation analysis revealed that AMPKα1 of cardiomyocytes was phosphorylated by A-769662.

Significance

This is the first study illustrating that activation of AMPK plays a significant role in mediating the contractile function of cardiomyocytes using transgenic animal models. AMPK activator facilitates the contractility of cardiomyocytes via activating AMPKα1 catalytic subunit. The phosphorylation of cTnI by AMPK could be a factor attributing to the regulation of contractility of cardiomyocytes.     

Atrial Natriuretic Peptide Regulates Ca2+ Channel in Early Developmental Cardiomyocytes

Background

Cardiomyocytes derived from murine embryonic stem (ES) cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP) in regulation of membrane potentials and Ca²⁺ currents has not been investigated in developmental cardiomyocytes.

Methodology/Principal Findings

We investigated the role of ANP in regulating L-type Ca²⁺ channel current (I_CaL) in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs) in early developmental stage (EDS) cardiomyocytes, embryonic bodies (EB) as well as whole embryo hearts. ANP exerted an inhibitory effect on basal I_CaL in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS) cells. However, after stimulation of I_CaL by isoproterenol (ISO) in LDS cells, ANP inhibited the response in about 70% cells. The depression of I_CaL induced by ANP was not affected by either Nω, Nitro-L-Arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG) selective inhibitor, in either EDS and LDS cells; whereas depression of I_CaL by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl) adenine (EHNA), a selective inhibitor of type 2 phosphodiesterase(PDE2) in most cells tested.

Conclusion/Significances

Taken together, these results indicate that ANP induced depression of action potentials and I_CaL is due to activation of particulate guanylyl cyclase (GC), cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3′, 5′–cyclic monophophate (cAMP)–cAMP-dependent protein kinase (PKA) in early cardiomyogenesis.     

Fluvoxamine rescues mitochondrial Ca transport and ATP production through σ1-receptor in hypertrophic cardiomyocytes

Aims

We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ₁-receptor (σ₁R), ameliorates cardiac hypertrophy and dysfunction via σ₁R stimulation. Although σ₁R on non-cardiomyocytes interacts with the IP₃ receptor (IP₃R) to promote mitochondrial Ca^2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main methods

Here we performed Ca^2 + imaging and measured ATP production to define the role of σ₁Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca^2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key finding

These cardiomyocytes exhibited imbalances in expression levels of σ₁R and IP₃R and impairments in both phenylephrine-induced mitochondrial Ca^2 + mobilization from the SR and ATP production. Interestingly, σ₁R stimulation with fluvoxamine rescued impaired mitochondrial Ca^2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ₁R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ₁Rs suppressed intracellular Ca^2 + mobilization through IP₃Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance

These results suggest that σ₁R stimulation with fluvoxamine promotes SR-mitochondrial Ca^2 + transport and mitochondrial ATP production, whereas σ₁R stimulation suppresses intracellular Ca^2 + overload through IP₃Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ₁R agonists including fluvoxamine.     

Fructose modulates cardiomyocyte excitation-contraction coupling and Ca²⁺ handling in vitro

Background

High dietary fructose has structural and metabolic cardiac impact, but the potential for fructose to exert direct myocardial action is uncertain. Cardiomyocyte functional responsiveness to fructose, and capacity to transport fructose has not been previously demonstrated.

Objective

The aim of the present study was to seek evidence of fructose-induced modulation of cardiomyocyte excitation-contraction coupling in an acute, in vitro setting.

Methods and Results

The functional effects of fructose on isolated adult rat cardiomyocyte contractility and Ca²⁺ handling were evaluated under physiological conditions (37°C, 2 mM Ca²⁺, HEPES buffer, 4 Hz stimulation) using video edge detection and microfluorimetry (Fura2) methods. Compared with control glucose (11 mM) superfusate, 2-deoxyglucose (2 DG, 11 mM) substitution prolonged both the contraction and relaxation phases of the twitch (by 16 and 36% respectively, p<0.05) and this effect was completely abrogated with fructose supplementation (11 mM). Similarly, fructose prevented the Ca²⁺ transient delay induced by exposure to 2 DG (time to peak Ca²⁺ transient: 2 DG: 29.0±2.1 ms vs. glucose: 23.6±1.1 ms vs. fructose +2 DG: 23.7±1.0 ms; p<0.05). The presence of the fructose transporter, GLUT5 (Slc2a5) was demonstrated in ventricular cardiomyocytes using real time RT-PCR and this was confirmed by conventional RT-PCR.

Conclusion

This is the first demonstration of an acute influence of fructose on cardiomyocyte excitation-contraction coupling. The findings indicate cardiomyocyte capacity to transport and functionally utilize exogenously supplied fructose. This study provides the impetus for future research directed towards characterizing myocardial fructose metabolism and understanding how long term high fructose intake may contribute to modulating cardiac function.     

Protein Kinase C-mediated Phosphorylation and Activation of PDE3A Regulate cAMP Levels in Human Platelets

The elevation of [cAMP]_i is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Stimulation of platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient phosphorylation of PDE3A on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹², in parallel with the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation and activation of PDE3A required the activation of PKC, but not of PI3K/PKB, mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent manner. This was further supported by the finding that IGF-1, which strongly activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE₁ and forskolin-induced phosphorylation of Ser³¹² and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE₁-evoked cAMP accumulation by thrombin required both G_i and PKC activation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹² leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding.Upon vascular injury, platelets adhere to the newly exposed subintimal collagen and undergo activation leading to platelet spreading to cover the damaged region and release of thrombogenic factors such as ADP and thromboxane A₂. In addition, platelets are activated by thrombin, which is generated as a result of activation of the coagulation pathway, and stimulates platelets by cleaving the protease-activated receptors (PAR),² PAR-1 and PAR-4. The final common pathway is the exposure of fibrinogen binding sites on integrin α_IIbβ₃ resulting in platelet aggregation and thrombus formation.Thrombin-mediated cleavage of PARs leads to activation of phospholipase C β (PLC), hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and a subsequent increase in [Ca²⁺]_i and activation of protein kinase C (PKC). Protein kinase C contributes to platelet activation both directly, through affinity regulation of the fibrinogen receptor, integrin α_IIbβ₃ (1), and indirectly by enhancing degranulation (2). Thrombin also stimulates activation of PI 3-kinases and subsequent generation of PI (3, 4, 5) trisphosphate and PI (3, 4) bisphosphate (3), which recruit protein kinase B (PKB) to the plasma membrane where it becomes phosphorylated and activated.Platelet activation is opposed by agents that raise intracellular 3′-5′-cyclic adenosine monophosphate ([cAMP]_i). cAMP is a powerful inhibitory second messenger that down-regulates platelet function by interfering with Ca²⁺ homeostasis, degranulation and integrin activation (4). Synthesis of cAMP is stimulated by mediators such as prostaglandin I₂ (PGI₂), which bind to G_s-coupled receptors leading to activation of adenylate cyclase (AC). This inhibitory pathway is opposed by thrombin, which inhibits the elevation of cAMP indirectly via autocrine activation of the G_i-coupled ADP receptor P2Y₁₂. cAMP signaling is terminated by hydrolysis to biologically inert 5′-AMP by 3′-phosphodiesterases. Platelets express two cAMP phosphodiesterase isoforms, cGMP-stimulated PDE2 and cGMP-inhibited PDE3A. PDE3A is the most abundant isoform in platelets and has a ∼250-fold lower K_m for cAMP than PDE2 (4). As a consequence of these properties, PDE3A exerts a greater influence on cAMP homeostasis, particularly at resting levels. The importance of PDE3A in platelet function is further emphasized by the finding that the PDE3A inhibitors cilostamide and milrinone raise basal cAMP levels and strongly inhibit thrombin-induced platelet activation (5). Furthermore, PDE3A^-/- mice demonstrate increased resting levels of platelet cAMP and are protected against a model of pulmonary thrombosis (6). In contrast, the PDE2 inhibitor EHNA has no significant effect on cAMP levels and platelet aggregation (7, 8). The activity of PDE3A is therefore essential to maintain low equilibrium levels of cAMP and determine the threshold for platelet activation (7).Like its paralogue PDE3B, it has recently become clear that PDE3A activity can be positively regulated by phosphorylation in platelets and human oocytes (9, 10). There is some evidence that PKB may be involved in this regulation, although the phosphorylation sites are poorly characterized. In contrast, phosphorylation of PDE3A in HeLa cells was stimulated by phorbol esters and blocked by inhibitors of PKC (11). In this study, we aimed to identify the signaling pathways and phosphorylation sites that are involved in regulation of platelet PDE3A. Here, we show strong evidence that PKC, and not PKB, is involved in agonist-stimulated PDE3A phosphorylation on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹², leading to an increase in PDE3A activity, 14-3-3 binding and modulation of intracellular cAMP levels.     

Defective sarcoplasmic reticulum–mitochondria calcium exchange in aged mouse myocardium

Mitochondrial alterations are critically involved in increased vulnerability to disease during aging. We investigated the contribution of mitochondria–sarcoplasmic reticulum (SR) communication in cardiomyocyte functional alterations during aging. Heart function (echocardiography) and ATP/phosphocreatine (NMR spectroscopy) were preserved in hearts from old mice (>20 months) with respect to young mice (5–6 months). Mitochondrial membrane potential and resting O₂ consumption were similar in mitochondria from young and old hearts. However, maximal ADP-stimulated O₂ consumption was specifically reduced in interfibrillar mitochondria from aged hearts. Second generation proteomics disclosed an increased mitochondrial protein oxidation in advanced age. Because energy production and oxidative status are regulated by mitochondrial Ca²⁺, we investigated the effect of age on mitochondrial Ca²⁺ uptake. Although no age-dependent differences were found in Ca²⁺ uptake kinetics in isolated mitochondria, mitochondrial Ca²⁺ uptake secondary to SR Ca²⁺ release was significantly reduced in cardiomyocytes from old hearts, and this effect was associated with decreased NAD(P)H regeneration and increased mitochondrial ROS upon increased contractile activity. Immunofluorescence and proximity ligation assay identified the defective communication between mitochondrial voltage-dependent anion channel and SR ryanodine receptor (RyR) in cardiomyocytes from aged hearts associated with altered Ca²⁺ handling. Age-dependent alterations in SR Ca²⁺ transfer to mitochondria and in Ca²⁺ handling could be reproduced in cardiomyoctes from young hearts after interorganelle disruption with colchicine, at concentrations that had no effect in aged cardiomyocytes or isolated mitochondria. Thus, defective SR–mitochondria communication underlies inefficient interorganelle Ca²⁺ exchange that contributes to energy demand/supply mistmach and oxidative stress in the aged heart.Age is the main independent risk factor for cardiovascular morbidity and mortality.¹ It increases heart vulnerability to cardiac diseases as well as the severity of their clinical manifestations, and reduces the efficacy of cardioprotective interventions.² At the cellular level, some of the structural and functional age-dependent changes resemble those of failing cardiac myocytes.^{3, 4} Specifically, disturbed Ca²⁺ homeostasis and excitation–contraction coupling,⁵ as well as deficient mitochondrial energetics⁶ and excessive ROS production,⁷ have been consistently reported in senescent cardiomyocytes. These subcellular alterations likely contribute to the reduced adaptive capacity to stress (exercise, β-adrenergic stimulation) and increased vulnerability to disease of the aged hearts.In cardiac cells, electrochemical coupling and metabolic adaptations are based upon the coordination between sarcoplasmic reticulum (SR) and mitochondria tightly interconnected forming an interface to support local ionic exchange and signal transduction in a beat-to-beat basis.⁸ This privileged interorganelle communication facilitates mitochondrial ATP transport for SR Ca²⁺ cycling and ensures energy replenishment by reciprocal Ca²⁺ and ADP exchange. Ca²⁺ is taken up by mitochondria using a low-affinity uniporter whose activity is driven by the elevated Ca²⁺ concentration in the microenvironment present around ryanodine receptors (RyR).⁹ Indeed, the kinetics of mitochondrial Ca²⁺ uptake is more dependent on the concentration of Ca²⁺ at the SR–mitochondria contact points than on bulk cytosolic Ca²⁺ concentration.⁸ Mitochondrial Ca²⁺ uptake allows energy supply–demand matching through the activation of Krebs cycle dehydrogenases and electron transport chain activity, and at the same time it regulates the regeneration of Krebs-coupled antioxidative defenses (NAD(P)H).¹⁰Defective SR–mitochondria cross talk has been causally linked to the abnormal mitochondrial Ca²⁺ uptake in failing hearts and may underlie their increased oxidative stress.¹¹ Also, in diabetic cardiomyopathy, intracellular Ca²⁺ overload and depletion of energy stores appear to develop as a consequence of sequential SR–mitochondria dysfunction.¹² Atrial fibrillation has been associated with an increased fusion of mitochondria and a subsequent increased colocalization of giant mitochondria with SR, a subcellular remodeling process that contributes to the perpetuation of the arrhythmia.¹³ Because mitochondria are highly dynamic structures, some molecular links have been proposed to provide a stable physical interorganelle bridge^{14, 15} while others appear to facilitate direct tunneling of Ca²⁺ and other signaling mediators.¹⁶ In the present study, we hypothesized that aging may negatively impact on mitochondria–SR communication by mechanisms involving defective Ca²⁺ transmission, and we identified reduced physical interaction between RyR and mitochondrial voltage-dependent anion channel (VDAC) as the main responsible of this effect.