Myocyte Shortening Research Articles

Effects of GH Secretagogues on Contractility and Ca2+Homeostasis of Isolated Adult Rat Ventricular Myocytes

Ghrelin and its synthetic analogue hexarelin are specific ligands of GH secretagogue receptor (GHS-R) and induce a variety of cardiovascular protective and cardiac positive inotropic effects. The signaling system underlying immediate effects of both GHSs in cardiomyocytes remains undefined. In the present study, we investigated the immediate effects of GHSs on isolated ventricular myocyte shortening, intracellular Ca(2+) ([Ca(2+)](i)) transients, and the L-type Ca(2+) current (I(Ca,L)). Putative intracellular signalling cascades were studied with specific receptor and signalling blockers. In fresh isolated adult Wistar rat ventricular myocytes, GHSs produced a positive inotropic effect in a concentration-dependent manner and increased the amplitude of [Ca(2+)](i) transients and the I(Ca,L). The positive inotropic response was abolished by the GHS-R1a antagonist [D-Lys(3)]-GH-releasing peptide-6 (10 microm). GHS-induced increase in the I(Ca,L) was abolished by [D-Lys(3)]-GH-releasing peptide-6 and protein kinase C inhibitor, chelerythrine chloride (5 microm), but not by protein kinase A inhibitor, KT 5720 (10 microm). We conclude that hexarelin and ghrelin increase the I(Ca,L), through GHS-R1a receptor and protein kinase C signalling cascade, which contribute to its direct positive inotropic effect on cardiomyocytes.

Alterations of L-Type Calcium Current and Cardiac Function in CaMKIIδ Knockout Mice

Recent studies have highlighted important roles of CaMKII in regulating Ca(2+) handling and excitation-contraction coupling. However, the cardiac effect of chronic CaMKII inhibition has not been well understood. We have tested the alterations of L-type calcium current (I(Ca)) and cardiac function in CaMKIIdelta knockout (KO) mouse left ventricle (LV). We used the patch-clamp method to record I(Ca) in ventricular myocytes and found that in KO LV, basal I(Ca) was significantly increased without changing the transmural gradient of I(Ca) distribution. Substitution of Ba(2+) for Ca(2+) showed similar increase in I(Ba). There was no change in the voltage dependence of I(Ca) activation and inactivation. I(Ca) recovery from inactivation, however, was significantly slowed. In KO LV, the Ca(2+)-dependent I(Ca) facilitation (CDF) and I(Ca) response to isoproterenol (ISO) were significantly reduced. However, ISO response was reversed by beta2-adrenergic receptor (AR) inhibition. Western blots showed a decrease in beta1-AR and an increase in Ca(v)1.2, beta2-AR, and Galphai3 protein levels. Ca(2+) transient and sarcomere shortening in KO myocytes were unchanged at 1-Hz but reduced at 3-Hz stimulation. Echocardiography in conscious mice revealed an increased basal contractility in KO mice. However, cardiac reserve to work load and beta-adrenergic stimulation was reduced. Surprisingly, KO mice showed a reduced heart rate in response to work load or beta-adrenergic stimulation. Our results implicate physiological CaMKII activity in maintaining normal I(Ca), Ca(2+) handling, excitation-contraction coupling, and the in vivo heart function in response to cardiac stress.

NADPH oxidase-derived superoxide impairs calcium transients and contraction in aged murine ventricular myocytes

Since aging increases oxidative stress, we analyzed the contribution of reactive oxygen species (ROS) to the contractile dysfunction of aged ventricular myocytes and investigated whether short-term interference with ROS formation could normalize contractile performance. Isolated ventricular myocytes from young (2–4 months) and aged (24–26 months) male mice (C57BL/6) were used. We analyzed sarcomere shortening and calcium transients (Indo-1 fluorescence) of voltage clamped ventricular myocytes and myofilament ATPase activity (malachite green assay). Expression of calcium handling proteins (Western blots) and NADPH oxidase subunits (real-time PCR) was quantified, as well as NADPH oxidase activity (lucigenin chemiluminescence). We found that aged myocytes showed decelerated shortening/relengthening without changes in fractional shortening. Calcium transient decay was similarly decelerated, but the amplitude of calcium transients was increased with aging. Calcium sensitivity of myofilaments of aged myocytes was reduced. These age-dependent changes occurred without altered calcium handling protein expression but were reversed by the superoxide scavenger tiron. Aged myocytes showed increased NADPH oxidase expression and activity. Pharmacological inhibition of NADPH oxidase (diphenylene iodonium; apocynin) normalized age-dependent deceleration of shortening/relengthening. In summary, we show that increased superoxide formation by upregulated NADPH oxidase contributes significantly to age-dependent alterations in calcium handling and contractility of murine ventricular myocytes.

Hypoxia inducible factor-1 improves the negative functional effects of natriuretic peptide and nitric oxide signaling in hypertrophic cardiac myocytes

Aims Both natriuretic peptides and nitric oxide may be protective in cardiac hypertrophy, although their functional effects are diminished in hypertrophy. Hypoxia inducible factor-1 (HIF-1) may also protect in cardiac hypertrophy. We hypothesized that upregulation of HIF-1 would protect the functional effects of cyclic GMP (cGMP) signaling in hypertrophied ventricular myocytes. Main methods A cardiac hypertrophy model was created in mice by transverse aorta constriction. HIF-1 was increased by deferoxamine (150 mg/kg for 2 days). HIF-1α protein levels were examined. Functional parameters were measured (edge detector) on freshly isolated myocytes at baseline and after BNP (brain natriuretic peptide, 10 − 8 –10 − 7 M) or CNP (C-type natriuretic peptide, 10 − 8 –10 − 7 M) or SNAP (S-nitroso-N-acetyl-penicillamine, a nitric oxide donor, 10 − 6 –10 − 5 M) followed by KT5823 (a cyclic GMP-dependent protein kinase (PKG) inhibitor, 10 − 6 M). We also determined PKG expression levels and kinase activity. Key findings We found that under control conditions, BNP (− 24%), CNP (− 22%) and SNAP (− 23%) reduced myocyte shortening, while KT5823 partially restored function. Deferoxamine treated control myocytes responded similarly. Baseline function was reduced in the myocytes from hypertrophied heart. BNP, CNP, SNAP and KT5823 also had no significant effects on function in these myocytes. Deferoxamine restored the negative functional effects of BNP (− 22%), CNP (− 18%) and SNAP (− 19%) in hypertrophic cardiac myocytes and KT5823 partially reversed this effect. Additionally, deferoxamine maintained PKG expression levels and activity in hypertrophied heart. Significance Our results indicated that the HIF-1 protected the functional effects of cGMP signaling in cardiac hypertrophy through preservation of PKG.

Carbonylation of myosin heavy chains in rat heart during diabetes

Cardiac inotropy progressively declines during diabetes mellitus. To date, the molecular mechanisms underlying this defect remain incompletely characterized. This study tests the hypothesis that ventricular myosin heavy chains (MHC) undergo carbonylation by reactive carbonyl species (RCS) during diabetes and these modifications contribute to the inotropic decline. Male Sprague–Dawley rats were injected with streptozotocin (STZ). Fourteen days later the animals were divided into two groups: one group was treated with the RCS blocker aminoguanidine for 6 weeks, while the other group received no treatment. After 8 weeks of diabetes, cardiac ejection fraction, fractional shortening, left ventricular pressure development (+d P/d t) and myocyte shortening were decreased by 9%, 16%, 34% and 18%, respectively. Ca 2+- and Mg 2+-actomyosin ATPase activities and peak actomyosin syneresis were also reduced by 35%, 28%, and 72%. MHC-α to MHC-β ratio was 12:88. Mass spectrometry and Western blots revealed the presence of carbonyl adducts on MHC-α and MHC-β. Aminoguanidine treatment did not alter MHC composition, but it blunted formation of carbonyl adducts and decreases in actomyosin Ca 2+-sensitive ATPase activity, syneresis, myocyte shortening, cardiac ejection fraction, fractional shortening and +d P/d t induced by diabetes. From these new data it can be concluded that in addition to isozyme switching, modification of MHC by RCS also contributes to the inotropic decline seen during diabetes.

Alloxan reduces amplitude of ventricular myocyte shortening and intracellular Ca2+ without altering L-type Ca2+ current, sarcoplasmic reticulum Ca2+ content or myofilament sensitivity to Ca2+ in Wistar rats

Alloxan is widely used to induce diabetes mellitus in experimental animals. Recent studies have provided evidence that alloxan has direct actions on cardiac muscle contraction. The aim of this study was to further investigate the mechanisms underlying the effects of alloxan on ventricular myocyte shortening and intracellular Ca(2+) transport. Amplitude of myocyte shortening was reduced in a dose-dependent manner as the concentration of alloxan was increased in the range 10(-7)-10(-4) M. Amplitude of shortening was reduced (56.8 +/- 6.6%, n = 27) by 10(-6) M alloxan and was partially reversed during a 10 min washout. Amplitude of the Ca(2+) transient was also reduced (79.7 +/- 2.9%, n = 29) by 10(-6) M alloxan. Caffeine-evoked sarcoplasmic reticulum Ca(2+) release, fractional release of Ca(2+), assessed by comparing the amplitude of electrically evoked with that of caffeine-evoked Ca(2+) transients, and fura-2-cell length trajectory during the late stages of relaxation of myocyte twitch contraction were not significantly altered by alloxan. The amplitude of L-type Ca(2+) current was not altered by alloxan. Alterations in sarcoplasmic reticulum Ca(2+) transport, myofilament sensitivity to Ca(2+), and L-type Ca(2+) current do not appear to underlie the negative inotropic effects of alloxan.

Determinants of Loaded Shortening in Cardiac Myocytes

Ventricular performance is dictated by stroke volume, which ultimately depends on the extent of myocyte shortening during loaded contractions. We propose that the extent of loaded shortening is determined by the balance between two processes: (i) Ca2+-cross-bridge-induced cooperative activation of the thin filament and (ii) shortening-induced cooperative deactivation of the thin filament. Accordingly, any modulator that augments contractility (i.e., stroke volume) should favor process (i) and diminish process (ii). Since β-adrenergic stimulation is known to increase contractility, we tested whether PKA (the myofibrillar ligand of β-adrenergic signaling) would increase cooperative activation and diminish shortening-induced deactivation in rat permeabilized cardiac myocytes during submaximal Ca2+ activations. Regarding cooperative activation, PKA increased the slope of tension-pCa relationships (nH = 3.85 ± 0.09 before versus nH = 5.03 ± 0.71 after PKA). PKA also slowed rates of force redevelopment, increased the transient force overshoot after a slack-restretch maneuver, and increased the rate and amplitude of spontaneous oscillatory contractions (SPOCs); all of which are consistent with greater cooperative activation of the thin filament. Regarding cooperative deactivation, PKA increased the curvature of myocyte length traces during lightly loaded shortening (kshortening = 6.41 ± 0.28 before versus kshortening = 9.45 ± 0.53 after PKA) and steepened sarcomere length-tension relationships; both of which implicate enhanced (rather than diminished) shortening-induced cooperative deactivation. Taken together, PKA-induced myofibrillar phosphorylation appears to augment both Ca2+-cross-bridge-induced cooperative activation of the thin filament and (ii) shortening-induced cooperative deactivation. Greater cooperative activation should lead to more cycling cross-bridges, which would speed loaded shortening against a given afterload. On the other hand, greater shortening-induced cooperative deactivation may be necessary to help accelerate relaxation and assist diastolic filling in the face of shorter systolic and diastolic times in the presence of higher heart rates induced by β-adrenergic stimulation.

Ultrastructural and Functional Remodeling of the Coupling Between Ca 2+ Influx and Sarcoplasmic Reticulum Ca 2+ Release in Right Atrial Myocytes From Experimental Persistent Atrial Fibrillation

Persistent atrial fibrillation (AF) has been associated with structural and electric remodeling and reduced contractile function. To unravel mechanisms underlying reduced sarcoplasmic reticulum (SR) Ca(2+) release in persistent AF. We studied cell shortening, membrane currents, and [Ca(2+)](i) in right atrial myocytes isolated from sheep with persistent AF (duration 129+/-39 days, N=16), compared to matched control animals (N=21). T-tubule density, ryanodine receptor (RyR) distribution, and local [Ca(2+)](i) transients were examined in confocal imaging. Myocyte shortening and underlying [Ca(2+)](i) transients were profoundly reduced in AF (by 54.8% and 62%, P<0.01). This reduced cell shortening could be corrected by increasing [Ca(2+)](i). SR Ca(2+) content was not different. Calculated fractional SR Ca(2+) release was reduced in AF (by 20.6%, P<0.05). Peak Ca(2+) current density was modestly decreased (by 23.9%, P<0.01). T-tubules were present in the control atrial myocytes at low density and strongly reduced in AF (by 45%, P<0.01), whereas the regular distribution of RyR was unchanged. Synchrony of SR Ca(2+) release in AF was significantly reduced with increased areas of delayed Ca(2+) release. Propagation between RyR was unaffected but Ca(2+) release at subsarcolemmal sites was reduced. Rate of Ca(2+) extrusion by Na(+)/Ca(2+) exchanger was increased. In persistent AF, reduced SR Ca(2+) release despite preserved SR Ca(2+) content is a major factor in contractile dysfunction. Fewer Ca(2+) channel-RyR couplings and reduced efficiency of the coupling at subsarcolemmal sites, possibly related to increased Na(+)/Ca(2+) exchanger, underlie the reduction in Ca(2+) release.

Calcineurin and cardiac function: is more or less better for the heart?

calcineurin (cn) is a Ca2+-calmodulin-activated serine-threonine phosphatase that is ubiquitously expressed and plays an important role in transducing Ca2+-dependent signals. Cn is a heterodimer composed of calmodulin binding catalytic subunit A (CnA) and a Ca2+ binding regulatory subunit B (CnB) ([

Effects of varying intensity exercise on shortening and intracellular calcium in ventricular myocytes from streptozotocin (STZ)-induced diabetic rats

Effects of varying intensity exercise on shortening and intracellular calcium in ventricular myocytes from streptozotocin (STZ)-induced diabetic rats

The effects of heavy long-term exercise on ventricular myocyte shortening and intracellular Ca 2+ in streptozotocin-induced diabetic rat

Objective This study investigated whether exercise training, initiated at the onset of diabetes, could preserve the contractile properties of ventricular myocytes. Research Design and Methods The effects of a heavy exercise training program on shortening and intracellular Ca 2+ in unloaded ventricular myocytes from streptozotocin (STZ)-induced diabetic rats were examined. Animals were divided into four groups: control sedentary (CS), diabetic sedentary (DS), control heavy exercise (CHE), and diabetic heavy exercise (DHE). Exercise protocol: 5×60 min/week, 18 m/min, 5% gradient. Exercise training began 1 week after STZ treatment and continued for 12–23 (mean 17.5) weeks. Results Diabetes induced prolongation of time-to-peak (TPK) shortening (124±2 ms in DS compared to 97±2 ms in CS rats), which was further increased by exercise (133±3 ms in DHE and 112±2 ms in CHE myocytes). Diabetes had no significant effects on time-to-half (THALF) relaxation of shortening (61±2 ms in DS compared to 56±2 ms in CS myocytes). Exercise induced significant prolongation of THALF in control (66±3 ms) but not in diabetic (69±3 ms) myocytes. Diabetes, though not exercise, significantly prolonged TPK (76±3 ms in DS compared to 64±2 ms in CS) and THALF recovery (160±5 ms in DS compared to 118±4 ms in CS) of the Ca 2+ transient. Neither diabetes nor exercise had significant effects on the amplitude of myocyte shortening and the Ca 2+ transient. Conclusions Heavy long-term exercise alters the dynamics but not the amplitude of unloaded myocyte contraction in the STZ-induced diabetic rat.

The role of nitric oxide and reactive oxygen species in the positive inotropic response to mechanical stretch in the mammalian myocardium

The endothelial nitric oxide synthase (eNOS) has been implicated in the rapid (Frank–Starling) and slow (Anrep) cardiac response to stretch. Our work and that of others have demonstrated that a neuronal nitric oxide synthase (nNOS) localized to the myocardium plays an important role in the regulation of cardiac function and calcium handling. However, the effect of nNOS on the myocardial response to stretch has yet to be investigated. Recent evidence suggests that the stretch-induced release of angiotensin II (Ang II) and endothelin 1 (ET-1) stimulates myocardial superoxide production from NADPH oxidases which, in turn, contributes to the Anrep effect. nNOS has also been shown to regulate the production of myocardial superoxide, suggesting that this isoform may influence the cardiac response to stretch or ET-1 by altering the NO-redox balance in the myocardium. Here we show that the increase in left ventricular (LV) myocyte shortening in response to the application of ET-1 (10 nM, 5 min) did not differ between nNOS−/− mice and their wild type littermates (nNOS+/+). Pre-incubating LV myocytes with the NADPH oxidase inhibitor, apocynin (100 μM, 30 min), reduced cell shortening in nNOS−/− myocytes only but prevented the positive inotropic effects of ET-1 in both groups. Superoxide production (O2−) was enhanced in nNOS−/− myocytes compared to nNOS+/+; however, this difference was abolished by pre-incubation with apocynin. There was no detectable increase in O2− production in ET-1 pre-treated LV myocytes. Inhibition of protein kinase C (chelerythrine, 1 μM) did not affect cell shortening in either group, however, protein kinase A inhibitor, PKI (2 μM), significantly reduced the positive inotropic effects of ET-1 in both nNOS+/+ and nNOS−/− myocytes. Taken together, our findings show that the positive inotropic effect of ET-1 in murine LV myocytes is independent of nNOS but requires NADPH oxidases and protein kinase A (PKA)-dependent signaling. These results may further our understanding of the signaling pathways involved in the myocardial inotropic response to stretch.

J014 Simultaneous recordings of cell shortening and camp or calcium transients reveal differential regulation of cardiac contractility by specific phosphodiesterases

Multiple cyclic nucleotide phosphodiesterases (PDEs) belonging to four families (PDE1 to PDE4) hydrolyze cAMP in cardiac cells, but the functional significance of this diversity is not well understood. The goal of this study was to characterize the involvement of different PDEs in excitation-contraction coupling in cardiomyocytes. For this, sarcomere shortening and Ca2+ transients were recorded simultaneously in rat ventricular myocytes field stimulated at 0.5 Hz with an IonOptix system. Selective inhibition of PDE2 with Bay 60-7550 (Bay, 100 nM) or PDE4 with Ro-201724 (Ro, 10μM) had no effect on basal cell contraction, whereas selective inhibition of PDE3 with cilostamide (Cil, 1μM) or β-adrenergic stimulation with isoprenaline (Iso, 1nM) increased myocyte shortening. Inhibition of PDE4 potentiated the response to Cil and Iso, showing that PDE4 becomes important when cAMP is prestimulated. Similar results were obtained on Ca2+ transients. cAMP measurements by FRET in beating cardiomyocytes indicate that Iso strongly increases cAMP levels. Effects of selective PDE inhibitors are under investigation. These results show that PDE2, PDE3 and PDE4 differentially regulate excitation-contraction coupling in cardiomyocytes.

Dofetilide Enhances the Contractility of Rat Ventricular Myocytes via Augmentation of Na+–Ca2+ Exchange

Dofetilide (DOF), a novel Class III antiarrhythmic drug, prolongs the action potential duration (APD) and shows a positive inotropic effect in guinea pig papillary muscle. The present experiments were designed to study the positive inotropic effect of DOF on rat ventricle and explore its possible mechanism(s). Hearts from male Wistar rats (260-320 g) were divided into five groups and perfused in Langendorff mode. Ventricular myocytes were enzymatically isolated from male Wistar rats. Whole-cell voltage-clamping technique was used to test the Na(+)-Ca(2+) exchange (NCE) current (I(NCX)); Calcium transients and cell shortening provoked by field stimulation or using calcium current command waveform were observed synchronously with an ionic imaging system. DOF (0.03-1.0 microM) increased left ventricular function in isolated rat hearts in a concentration-dependent manner. DOF (0.03-1.0 microM) also concentration-dependently increased both inward and outward I (NCX) in isolated rat ventricular cells. The EC(50) values of DOF were 0.149 microM for the inward I(NCX) and 0.249 microM for outward I(NCX), respectively. DOF 0.2 microM significantly enhanced Ca(2+) transient and cell shortening in single rat ventricular myocytes driven by field electric stimulation. When the patch clamp system was connected to the ionic imaging system, Ca(2+) current (I(Ca)), Ca(2+) transient and cell shortening amplitude in a same cell were recorded synchronously. Application of DOF 0.2 microM had no effect on I(Ca), but significantly increased Ca(2+) transient and cell shortening. NCX inhibitor KB-R7943 0.6 microM significantly depressed the effects of DOF on Ca(2+) transient and cell shortening. We conclude that DOF enhanced contractility of rat ventricular myocytes. The enhancement of NCE may be involved in the positive inotropic action of DOF.

Structural and Functional Characterization of Cardiac Troponin T Mutations in the TNT1 Domain That Cause Familial Hypertrophic Cardiomyopathy

FHC is a primary cardiac muscle disorder that is one of the most common causes of sudden death in young people. FHC “hotspot” mutations at residue 92 in cardiac troponin T (cTnT) flank the proposed α-helical TNT1 tail domain whose flexibility has been suggested to be important in normal protein-protein interactions within the thin filament. Through Molecular Dynamics (MD) simulations, we showed that FHC mutations Arg92Leu, Arg92Trp, and Arg92Gln cause local α-helical structural changes and increased flexibility at a critical hinge region 18 Angstroms distant from the mutation. We have extended this MD analysis via the use of a self-defined coordinate to measure localized bends in the helix and found that forces acting on this bending coordinate are lower in mutants than wildtype. This quantitatively suggests a less restrictive bending motion in mutants explaining the increased flexibility of the hinge region. To determine how primary biophysical changes induced by these mutations cause complex cardiomyopathies we hypothesize that flexibility alterations and changes in force within compaction-expansion regions in mutational segments lead to electrostatic perturbations, possibly interfering with cTnT-TM complex formation and thin filament function. In vitro motility assays with wildtype cTnT and hotspot FHC-cTnT mutants are in progress to directly correlate predicted alterations in electrostatic properties with resultant functional changes. Moreover, contractile and Ca2+ transient measurements on isolated myocytes address downstream myocellular responses to the mutation's primary perturbation on structure and function. Data showed normal percent shortening in Arg92Leu myocytes while Arg92Trp percent shortening was significantly impaired compared to Non-Tg (4.740 + 1.165 vs. 6.971 + 2.098, p<0.001). Completion of these studies will directly address the links between thin filament structure/function, downstream myocellular responses, and resultant distinct cardiovascular phenotypes.

Cardiac Ca2+ Release Channel/RyR2 -Molecular Mechanism Of Green Tea Extract epigallocatechin-3-gallate

Consumption of green tea has been found to closely correlate with a lower risk of heart disease and reduced cardiovascular mortality. Extracted from green leaves, a group of catechin polyphenols appears to be responsible for these beneficial cardiovascular effects. Studies have shown that epigallocatechin-3-gallate (EGCG), one of the most abundant and potent catechins, can modulate myocardial contractility through some unknown mechanisms. Recent evidence suggests a role for Na+/H+ and Na+/Ca2+ exchangers in the ionotropic effects of EGCG in rat heart. Here we report that cardiac Ca2+ release channel - RyR2 serves as a possible molecular target of EGCG that may be responsible for amplified Ca2+ transients, enhanced ventricular contractility and increased cell shortening in cardiac myocytes. In single isolated mouse ventricular myocytes EGCG (5μM) increased peak systolic Ca2+ transient amplitude, increased decay of the Ca2+ transient, increased fractional shortening and increased contraction and relaxation velocities. To assess the direct interaction of EGCG with RyR2, both purified and membrane-bound RyR2 single channels were reconstituted in planar lipid bilayers. In the presence of 0.5μM EGCG, the open probability (Po) increased from Po=0.012 in the control state to Po=0.387. Using [3H]ryanodine as a probe to assess channel conformational states, our data further indicate that (1) EGCG dose-dependently enhanced junctional SR membrane-bound RyR2 channel activity and (2) EGCG increased Ca2+-induced Ca2+ activation of RyR2 (EC50 = 7.0 and 1.7 μM for control and EGCG, respectively). In summary, our data are consistent with previous reports for positive inotropic and lusitropic effects by EGCG on heart cells. In addition, our data clearly supports a role of EGCG in modulating cardiac E-C coupling through interacting with RyR2 channel complex. Supported by K01 AR51519 to GR and P01 AR52354.

Simultaneous Recordings of Cell Shortening and cAMP or Calcium Transients Reveal Differential Regulation of Cardiac Contractility by Specific Phosphodiesterases

Multiple cyclic nucleotide phosphodiesterases (PDEs) belonging to four families (PDE1 to PDE4) hydrolyze cAMP in cardiac cells, but the functional significance of this diversity is not well understood. The goal of this study was to characterize the involvement of different PDEs in excitation-contraction coupling in cardiomyocytes. For this, sarcomere shortening and Ca2+ transients were recorded simultaneously in rat ventricular myocytes field stimulated at 0.5 Hz with an IonOptix system. As shown in the figure, selective inhibition of PDE2 with Bay 60-7550 (Bay, 100 nM) or PDE4 with Ro-201724 (Ro, 10 μM) had no effect on basal cell contraction, whereas selective inhibition of PDE3 with cilostamide (Cil, 1 μM) or β-adrenergic stimulation with isoprenaline (Iso, 1 nM) increased myocyte shortening. Inhibition of PDE4 potentiated the response to Cil and Iso, showing that PDE4 becomes important when cAMP is prestimulated. Similar results were obtained on Ca2+ transients. cAMP measurements by FRET in beating cardiomyocytes indicate that Iso strongly increases cAMP levels. Effects of selective PDE inhibitors are under investigation. These results show that PDE2, PDE3 and PDE4 differentially regulate excitation-contraction coupling in cardiomyocytes.

The direct effects of streptozotocin and alloxan on contractile function in rat heart

Streptozotocin (STZ) and alloxan (ALX) are widely used to induce diabetes mellitus in experimental animals. The direct effects of STZ and ALX on the amplitude and time course of ventricular myocyte shortening and on cardiac action potentials were investigated. STZ and ALX (10 −5 M) were dissolved in normal Tyrode (NT), maintained at pH 7.4 and 37 °C and stored for either 15 or 60–120 min. Both compounds reduced the amplitude of myocyte shortening. Compared to NT the amplitude of shortening was 34.7 ± 5.0% and 35.2 ± 6.8% with STZ and 41.0 ± 5.5% and 37.3 ± 5.7% with ALX stored for 15 and 60–120 min, respectively. During a 10 min NT washout STZ myocytes recovered to 56.2 ± 8.3% and 60.5 ± 8.2% and ALX myocytes recovered to 88.9 ± 10.0% and 83.7 ± 9.9% after storage of compounds for 15 and 60–120 min, respectively. Perfusion of the whole heart with ALX induced bradycardia but had no effects on the duration of action potential repolarization at 50% and 70% from peak action potential. The negative inotropic effects of STZ and ALX were not altered by storage. The results suggest that some of the effects on heart reported in STZ- and ALX-induced diabetes may be partly attributed to direct action of these compounds.

Testosterone modulates cardiomyocyte Ca2+ 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.

Image processing techniques for assessing contractility in isolated adult cardiac myocytes.

We describe a computational framework for the comprehensive assessment of contractile responses of enzymatically dissociated adult cardiac myocytes. The proposed methodology comprises the following stages: digital video recording of the contracting cell, edge preserving total variation-based image smoothing, segmentation of the smoothed images, contour extraction from the segmented images, shape representation by Fourier descriptors, and contractility assessment. The different stages are variants of mathematically sound and computationally robust algorithms very well established in the image processing community. The physiologic application of the methodology is evaluated by assessing overall contraction in enzymatically dissociated adult rat cardiocytes. Our results demonstrate the effectiveness of the proposed approach in characterizing the true, two-dimensional, “shortening” in the contraction process of adult cardiocytes. We compare the performance of the proposed method to that of a popular edge detection system in the literature. The proposed method not only provides a more comprehensive assessment of the myocyte contraction process but also can potentially eliminate historical concerns and sources of errors caused by myocyte rotation or translation during contraction. Furthermore, the versatility of the image processing techniques makes the method suitable for determining myocyte shortening in cells that usually bend or move during contraction. The proposed method can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease modeling, transgeneity, or other common applications to mammalian cardiocytes.