Maximal Developed Tension Research Articles

X-ray Diffraction Analysis to Explore Molecular Traces of Eccentric Contraction on Rat Skeletal Muscle Parallelly Evaluated with Signal Protein Phosphorylation Levels.

We performed X-ray diffraction analyses on rat plantaris muscle to determine if there are strain-specific structural changes at the molecular level after eccentric contraction (ECC). ECC was elicited in situ by supramaximal electrical stimulation through the tibial nerve. One hour after a series of ECC sessions, the structural changes that remained in the sarcomere were evaluated using X-ray diffraction. Proteins involved in cell signaling pathways in the muscle were also examined. ECC elicited by 100, 75, and 50 Hz stimulation respectively developed peak tension of 1.34, 1.12 and 0.79 times the isometric maximal tetanus tension. The series of ECC sessions phosphorylated the forkhead box O proteins (FoxO) in a tension-time integral-dependent manner, as well as phosphorylated the mitogen-activated protein kinases (MAPK) and a protein in the mammalian target of rapamycin (mTOR) pathway in a maximal tension dependent manner. Compared to isometric contractions, ECC was more efficient in phosphorylating the signaling proteins. X-ray diffraction revealed that the myofilament lattice was preserved even after intense ECC stimulation at 100 Hz. Additionally, ECC < 75 Hz preserved the molecular alignment of myoproteins along the myofilaments, while 75-Hz stimulation induced a slight but significant decrease in the intensity of meridional troponin reflection at 1/38 nm−1, and of myosin reflection at 1/14.4 nm−1. These two reflections demonstrated no appreciable decrease with triple repetitions of the standard series of ECC sessions at 50 Hz, suggesting that the intensity decrease depended on the instantaneous maximal tension development rather than the total load of contraction, and was more likely linked with the phosphorylation of MAPK and mTOR signaling proteins.

AT1Receptor Blockade Improves Myocardial Functional Performance in Obesity.

ResumoFundamento A obesidade tem sido associada com ativação crônica do sistema renina-angiotensina-aldosterona e importantes alterações no desempenho cardíaco.Objetivo Avaliar a influência do bloqueio de receptores de angiotensina-II do tipo 1 (AT1) sobre a morfologia e desempenho cardíaco de ratos obesos por dietaMétodos Ratos Wistar (n=48) foram submetidos a dieta controle (2,9 kcal/g) ou hiperlipídica (3,6 kcal/g) durante 20 semanas. Após a 16ª semana, foram distribuídos em quatro grupos: Controle (CO), Obeso (OB), Controle Losartan (CL) e Obeso Losartan (OL). CL e OL receberam losartan (30 mg/kg/dia) na água durante quatro semanas. Posteriormente, foram analisadas composição corporal, pressão arterial sistólica (PAS) e ecocardiograma. A função de músculos papilares foi avaliada em situação basal com concentração de cálcio ([Ca2+]o) de 2,50 mM e após manobras inotrópicas: potencial pós-pausa (PPP), elevação da [Ca2+]o e durante estimulação beta-adrenérgica com isoproterenol. A análise dos resultados foi feita por meio de Two-Way ANOVA e teste de comparações apropriado. O nível de significância considerado foi de 5%.Resultados Embora a alteração da PAS não tenha se mantido ao final do experimento, a obesidade se associou com hipertrofia cardíaca e maior velocidade de encurtamento da parede posterior do ventrículo esquerdo.No estudo de músculos papilares em condição basal, CL mostrou menor velocidade máxima de variação negativa da tensão desenvolvida (-dT/dt) do que CO. O PPP de 60s promoveu menor -dT/dt e pico de tensão desenvolvida (TD) em OB e CL, comparados ao CO, e maior variação relativa de TD e velocidade máxima de variação positiva (+dT/dt) no OL em relação a CL e OB. Sob 1,5, 2,0 e 2,5mM de [Ca2+]o, o grupo OL exibiu maior -dT/dt do que CL.Conclusão Losartan melhora a função miocárdica de ratos com obesidade induzida por dieta. (Arq Bras Cardiol. 2020; 115(1):17-28)

Increased Contractile Function of Human Saphenous Vein Grafts Harvested by "No-Touch" Technique.

Saphenous vein grafts are the most common conduits used for coronary artery bypass grafting (CABG); however, no more than 60% of vein grafts remain open after 10 years and graft failure is associated with poor clinical outcome. The “no-touch” harvesting technique—where a sheet of perivascular tissue is retained around the vein—improves graft patency to over 80% after 16 years of follow-up, but the mechanism for the improved patency rate is unclear. In this study, we investigated acute functional differences between vein grafts harvested conventionally and by “no-touch” technique and explored the importance of perivascular tissue for reducing surgical trauma, minimizing excessive distension, and releasing vasoactive paracrine factors. Segments of human saphenous veins were obtained from CABG surgery and their functional properties investigated by isometric and isobaric myography. We found a broad diameter-tension relationship for human saphenous veins, with peak capacity for active tension development at diameters corresponding to transmural pressures around 60 mmHg. Across the investigated transmural pressure range between 10 and 120 mmHg, maximal tension development was higher for “no-touch” compared to conventionally harvested saphenous veins. Contractile responses to serotonin, noradrenaline, and depolarization induced with elevated extracellular [K+] were significantly larger for saphenous veins harvested by “no-touch” compared to conventional technique. Conventional vein grafts are routinely pressurized manually in order to test for leaks; however, avoiding this distension procedure did not change the acute contractile function of the conventionally excised saphenous veins. In contrast, even though surgical removal of perivascular tissue during conventional harvesting was associated with a substantial decrease in force development, removal of perivascular tissue by careful dissection under a stereomicroscope only marginally affected contractile responses of veins harvested by “no-touch” technique. In conclusion, we show that saphenous veins harvested by “no-touch” technique have greater contractile capacity than veins harvested by conventional technique. The different capacity for smooth muscle contraction is not due to vasoactive substances released by the perivascular tissue. Instead, we propose that the larger tension development of saphenous veins harvested by “no-touch” technique reflects reduced surgical damage, which may have long-term consequences that contribute to the superior graft patency.

Regular exercise improves cardiac contractile activation by modulating MHC isoforms and SERCA activity in orchidectomized rats.

Data from the trial known as Testosterone in Older Men with Mobility Limitations (TOM) has indicated an association between testosterone administration and a greater risk for adverse cardiovascular events. We therefore propose that regular exercise is a cardioprotective alternative that prevents detrimental changes in contractile activation when a deficiency in male sex hormones exists. Ten-week-old orchidectomized (ORX) rats were subjected to a 9-wk treadmill running program at moderate intensity starting 1 wk after surgery. Although exercise-induced cardiac hypertrophy was observed both in rats that underwent ORX and sham surgery, regular exercise enhanced cardiac myofilament Ca(2+) sensitivity and myosin light-chain 2 phosphorylation only in rats that underwent a sham operation. Although the rats that had sham surgery and and given exercise exhibited no change in maximum developed tension, regular running prevented the suppression of maximum active tension in the hearts of ORX rats. Regular exercise also prevented a shift in myosin heavy chain (MHC) isoforms toward β-MHC, a reduction in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) activity, and an increase in SERCA sensitivity in the hearts of ORX rats. Neither SERCA content nor its modulating component, phospholamban (PLB), was altered by exercise in either sham-operated or ORX rats. However, decreases in the phosphorylated Thr(17) form of PLB and the phosphorylated Thr(287) form of Ca(2+)/calmodulin-dependent kinase II in the hearts of ORX rats were abolished after regular exercise. These results thus support the use of regular running as a cardioprotective alternative to testosterone replacement in hypogonadal conditions.

Coronary vascular function is improved in ischemic patients after continuous‐flow left ventricular assist device implantation (1071.8)

Continuous‐flow left ventricular assist devices (LVADs) are used in heart failure (HF) patients either as a bridge to transplantation or as a permanent (destination) therapy. HF is precipitated by myocardial infarction (MI) in most (ischemic; I) but not all (non‐ischemic; NI) patients. We hypothesized that coronary vascular dysfunction is greater in I (63±2 y; n=12) vs. NI (53±5 y; n=17) male HF patients, and that dysfunction is reversed by LVAD support. Arteries from a transmural biopsy of the left‐ventricle were obtained, and concentration‐response curves to bradykinin (BK, 10‐6 to 10‐10 M) and sodium nitroprusside (SNP, 10‐4 to 10‐9 M) were completed after vessels were precontracted to ~65% of maximal tension development. Maximal BK‐induced vasorelaxation was less (p&lt;0.05) in arteries from I (68±8%; n=21 arteries, 195±20 µm i.d.) vs. NI (87±3%; n=33 arteries, 213±17 µm i.d.) patients, while responses to SNP (~90%) were similar between groups. These findings indicate endothelial dysfunction is greater in I vs. NI patients at the time of LVAD implant. Next we assessed vascular reactivity in 6 I (58±6 y) and 4 NI (49±11 y) patients wherein samples were obtained at implant and explant (239±51 days later). Maximal BK‐induced vasorelaxation was greater (p&lt;0.05) in coronary arteries from I patients at explant (87±6%, n=14 arteries, 305±30 μm i.d.) vs. implant (53±11%, n=12 arteries, 204±33 μm i.d.). Maximal BK‐induced vasorelaxation was similar in coronary arteries from NI patients obtained at explant (79±8%, n=7 arteries, 232±21 μm i.d.) and implant (72±17%, n=5 arteries, 135±13 μm i.d.). Responses to SNP were similar (~93%) at implant and explant for I and NI patients. Our data demonstrate that endothelial dysfunction in patients with advanced ischemic cardiomyopathy is improved through LVAD support.

Eccentric exercise: mechanisms and effects when used as training regime or training adjunct

The aim of the current review is to discuss applications and mechanism of eccentric exercise in training regimes of competitive sports. Eccentric muscle work is important in most sports. Eccentric muscle contractions enhance the performance during the concentric phase of stretch-shortening cycles, which is important in disciplines like sprinting, jumping, throwing, and running. Muscles activated during lengthening movements can also function as shock absorbers, to decelerate during landing tasks or to precisely deal with high external loading in sports like alpine skiing. The few studies available on trained subjects reveal that eccentric training can further enhance maximal muscle strength and power. It can further optimize muscle length for maximal tension development at a greater degree of extension, and has potential to improve muscle coordination during eccentric tasks. In skeletal muscles, these functional adaptations are based on increases in muscle mass, fascicle length, number of sarcomeres, and cross-sectional area of type II fibers. Identified modalities for eccentric loading in athletic populations involve classical isotonic exercises, accentuated jumping exercises, eccentric overloading exercises, and eccentric cycle ergometry. We conclude that eccentric exercise offers a promising training modality to enhance performance and to prevent injuries in athletes. However, further research is necessary to better understand how the neuromuscular system adapts to eccentric loading in athletes.

Identifying cellular mechanisms of zinc-induced relaxation in isolated cardiomyocytes

We tested several molecular and cellular mechanisms of cardiomyocyte contraction-relaxation function that could account for the reduced systolic and enhanced diastolic function observed with exposure to extracellular Zn(2+). Contraction-relaxation function was monitored in isolated rat and mouse cardiomyocytes maintained at 37°C, stimulated at 2 or 6 Hz, and exposed to 32 μM Zn(2+) or vehicle. Intracellular Zn(2+) detected using FluoZin-3 rose to a concentration of ∼13 nM in 3-5 min. Peak sarcomere shortening was significantly reduced and diastolic sarcomere length was elongated after Zn(2+) exposure. Peak intracellular Ca(2+) detected by Fura-2FF was reduced after Zn(2+) exposure. However, the rate of cytosolic Ca(2+) decline reflecting sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) activity and the rate of Na(+)/Ca(2+) exchanger activity evaluated by rapid Na(+)-induced Ca(2+) efflux were unchanged by Zn(2+) exposure. SR Ca(2+) load evaluated by rapid caffeine exposure was reduced by ∼50%, and L-type calcium channel inward current measured by whole cell patch clamp was reduced by ∼70% in cardiomyocytes exposed to Zn(2+). Furthermore, ryanodine receptor (RyR) S2808 and phospholamban (PLB) S16/T17 were markedly dephosphorylated after perfusing hearts with 50 μM Zn(2+). Maximum tension development and thin-filament Ca(2+) sensitivity in chemically skinned cardiac muscle strips were not affected by Zn(2+) exposure. These findings suggest that Zn(2+) suppresses cardiomyocyte systolic function and enhances relaxation function by lowering systolic and diastolic intracellular Ca(2+) concentrations due to a combination of competitive inhibition of Ca(2+) influx through the L-type calcium channel, reduction of SR Ca(2+) load resulting from phospholamban dephosphorylation, and lowered SR Ca(2+) leak via RyR dephosphorylation. The use of the low-Ca(2+)-affinity Fura-2FF likely prevented the detection of changes in diastolic Ca(2+) and SERCA2a function. Other strategies to detect diastolic Ca(2+) in the presence of Zn(2+) are essential for future work.

N‐terminal region of cardiac myosin binding protein‐C impairs myofilament function

Cardiac myosin binding protein‐C (cMyBP‐C) is a thick filament protein that plays a critical role in the regulation of sarcomere structure and contractility. Previously, we demonstrated that the N‐terminal fragment of cMyBP‐C (C0‐C1f), released during ischemia‐reperfusion (I‐R) injury, is cytotoxic and significantly impairs contractile function. However, the mechanisms in which C0‐C1f impairs cardiomyocyte contractile function remain unknown. The hypothesis of this study is that C0‐C1f depresses cardiac myofilament function. The molecular mass of C0‐C1f was analyzed by top‐down mass spectrometry. The observed molecular weights were 30974.72 Da and 31989.20 Da for mouse and human species respectively. Permeabilized left ventricular (LV) muscle strips were incubated with purified C0‐C1f. Myofilament function was characterized by measuring active tension‐[Ca2+] relationships to determine changes in maximum tension development (tmax) and Ca2+ sensitivity. Tension development and ATP consumption were simultaneously measured to determine tension‐cost over a range of [Ca2+]. C0‐C1f significantly reduced myofilament Ca2+ sensitivity and tmax, increased tension‐cost, and increased the rate of tension redevelopment (ktr). These data contribute to the understanding of contractile dysfunction following I‐R injury and provide a novel therapeutic target for the treatment of heart failure.

Coronary arterial function is not impaired in patients following continuous‐flow left ventricular assist device implantation

Continuous‐flow left ventricular assist devices (LVADs) are used in advanced heart failure patients either to bridge them to transplantation or as a permanent‐destination therapy. We sought to determine whether chronic exposure to non‐pulsatile blood flow and acute increases in coronary perfusion pressure associated with LVAD implantation attenuate arterial function. A transmural biopsy of the left‐ventricle was obtained from seven male patients (52±6 years old) at the time of LVAD implant (pre) and 142±12 days later (post) upon LVAD explant. Arteries were dissected free and function was assessed using isometric tension techniques. First, Lmax tension was determined on each vessel. Next, contraction‐response curves to potassium chloride (KCl, 10–100 mM) were performed. To determine endothelium‐dependent and – independent vasorelaxation, respectively, concentration‐response curves to bradykinin (BK, 10−6 to 10−10 M) and sodium nitroprusside (SNP, 10−4 to 10−9 M) were completed on vessels precontracted to ~65% of maximal tension development. Maximal KCl‐evoked contraction (0.99±0.22 and 0.69±0.14 mg tension development/μm vessel length), maximal BK‐evoked vasorelaxation (64±13% and 80±7%), and maximal SNP‐evoked vasorelaxation (92±3% and 92±4%), was similar in arteries obtained from pre (n=13 arteries, 206±40 μm i.d.) and post (n=14 arteries, 288±24 μm i.d.) transmural biopsy samples, respectively. These preliminary data indicate that coronary vascular function is not attenuated by LVAD implantation.

Elevated rates of force development and MgATP binding in F764L and S532P myosin mutations causing dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a disease characterized by dilation of the ventricular chambers and reduced contractile function. We examined the contractile performance of chemically-skinned ventricular strips from two heterozygous murine models of DCM-causing missense mutations of myosin, F764L/+ and S532P/+, in an α-myosin heavy chain (MyHC) background. In Ca2+-activated skinned myocardial strips, the maximum developed tension in F764L/+ was only ~50% that of litter-mate controls (+/+). The F764L/+ also exhibited significantly reduced rigor stiffness, loaded shortening velocity and power output. Corresponding indices for S532P/+ strips were not different from controls. Manipulation of MgATP concentration in conjunction with measures of viscoelasticity, which provides estimates of myosin detachment rate 2πc, allowed us to probe the molecular basis of changes in crossbridge kinetics that occur with the myosin mutations. By examining the response of detachment rate to varying MgATP we found the rate of MgADP release was unaffected by the myosin mutations. However, MgATP binding rate was higher in the DCM groups compared to controls (422±109mM−1·s−1 in F764L/+, 483±74mM−1·s−1 in S532P/+ and 303±18mM−1·s−1 in +/+). In addition, the rate constant of force development, 2πb, was significantly higher in DCM groups compared to controls (at 5mM MgATP: 36.9±4.9s−1 in F764L/+, 32.9±4.5s−1 in S532P/+ and 18.2±1.7s−1 in +/+). These results suggest that elevated rates of force development and MgATP binding are features of cardiac myofilament function that underlie the development of DCM.

Diminished Contractile Responses of Isolated Conduit Arteries in Two Rat Models of Hypertension

Hypertension is accompanied by thickening of arteries, resulting in marked changes in their passive and active mechanical properties. The aim of this study was to demonstrate that the large conduit arteries from hypertensive individuals may not exhibit enhanced contractions in vitro, as is often claimed. Mechanical responses to vasoconstrictor stimuli were measured under isometric conditions using ring arterial segments isolated from spontaneously hypertensive rats, N(omega)-nitro-L-arginine methyl ester (L-NAME)-treated Wistar rats, and untreated Wistar rats serving as normotensive control. We found that thoracic aortas from both types of hypertensive rats had a greater sensitivity but diminished maximal developed tension in response to noradrenaline, when compared with that from normotensive rats. In superior mesenteric arteries, the sensitivity to noradrenaline was similar in all examined rat groups but in L-NAME-treated rats, these arteries exhibited decreased active force when stimulated with high noradrenaline concentrations, or with 100 mM KCl. These results indicate that hypertension leads to specific biomechanical alterations in diverse arterial types which are reflected in different modifications in their contractile properties.

Membrane acid‐base transporters modulate artery structure

Acid‐base transport and pH control modulate cell proliferation and migration with possible consequences for artery remodeling. Here, we used knockout mice for the Na+/H+‐exchanger NHE1 and Na+,HCO3−‐cotransporter NBCn1 to study their role in determining arterial structure. Measurements were performed using pH‐sensitive fluorophores, myography and stereological analyses. NHE1 mediated the Na+/H+‐exchange in mesenteric arteries, and knockout of NHE1 was associated with reduced media thickness and an equivalent inhibition of maximum active tension development. The reduced media thickness was due to a smaller smooth muscle cell volume and cross‐sectional area with no change in the number of smooth muscle cells per unit artery length. NBCn1 mediated the Na+,HCO3−‐cotransport in carotid arteries. Following partial carotid artery ligation, the inward remodeling (reduction in carotid artery diameter and increase in media area) in wild type mice was more than 50% attenuated in NBCn1 knockout mice. In conclusion, we show that the acid‐base transport proteins NBCn1 and NHE1 play an important role for determining artery structure. Supported by Danish Council for Independent Research and Danish Heart Foundation.

Expression of tropomyosin-κ induces dilated cardiomyopathy and depresses cardiac myofilament tension by mechanisms involving cross-bridge dependent activation and altered tropomyosin phosphorylation

Tropomyosin-kappa (TPM1-κ) is a newly discovered tropomyosin (TM) isoform that is exclusively expressed in the human heart and generated by an alternative splicing of the α-TM gene. We reported that TPM1-κ expression was increased in the hearts of patients with chronic dilated cardiomyopathy (DCM). To increase our understanding of the significance of this shift in isoform population, we generated transgenic (TG) mice expressing TPM1-κ in the cardiac compartment where TPM1-κ replaces 90% of the native TM. We previously showed that there was a significant inhibition of the ability of strongly bound cross-bridges to induce activation of TG myofilaments (Rajan et al., Circulation 121:410-418, 2010). Here, we compared the force-Ca(2+) relations in detergent extracted (skinned) fiber bundles isolated from non-transgenic (NTG) and TG-TPM1-κ hearts at two sarcomere lengths (SLs). Our data demonstrated a significant decrease in the Ca(2+) sensitivity of the myofilaments from TG-TPM1-κ hearts with no change in the maximum developed tension, length-dependent activation, and the ratio of ATPase rate to tension. There was also no difference in the affinity and cooperativity of Ca(2+)-binding to troponin in thin filaments reconstituted with either TPM1-κ or α-TM. We also compared protein phosphorylation in NTG and TG-TPM1-κ myofilaments. There was a decrease in the total phosphorylation of TPM1-κ compared to α-TM, but no significant change in other major sarcomeric proteins. Our results identify a novel mode of myofilament desensitization to Ca(2+) associated with a DCM linked switch in TM isoform population.

Knockout of insulin receptors in cardiomyocytes attenuates coronary arterial dysfunction induced by pressure overload

Ablating insulin receptors in cardiomyocytes causes subendocardial fibrosis and left ventricular (LV) dysfunction after 4 wk of transverse aortic constriction (TAC). To determine whether these maladaptive responses are precipitated by coronary vascular dysfunction, we studied mice with cardiomyocyte-restricted knock out of insulin receptors (CIRKO) and wild-type (WT) TAC mice before the onset of overt LV dysfunction. Two weeks of TAC produced comparable increases (P < 0.05 vs. respective sham) in heart weight/body weight (mg/g) in WT-TAC (8.03 ± 1.14, P < 0.05 vs. respective sham) and CIRKO-TAC (7.76 ± 1.25, P < 0.05 vs. respective sham) vs. WT-sham (5.64 ± 0.11) and CIRKO-sham (4.64 ± 0.10) mice. In addition, 2 wk of TAC were associated with similar LV geometry and function (echocardiography) and interstitial fibrosis (picrosirius red staining) in CIRKO and WT mice. Responses to acetylcholine (ACh), N(G)-monomethyl-L-arginine (l-NMMA), and sodium nitroprusside (SNP) were measured in coronary arteries that were precontracted to achieve ∼70% of maximal tension development using the thromboxane A(2) receptor mimetic U-46619 (∼3 × 10(-6) M). ACh-evoked vasorelaxation was absent in WT-TAC but was present in CIRKO-TAC albeit reduced relative to sham-operated animals. l-NMMA-evoked tension development was similar in vessels from CIRKO-TAC mice but was lower (P < 0.05) in WT-TAC animals vs. the respective sham-operated groups, and SNP-evoked vasorelaxation was similar among all mice. Thus estimates of stimulated and basal endothelial nitric oxide release were better preserved in CIRKO vs. WT mice in response to 2 wk of TAC. These findings indicate that maladaptive LV remodeling previously observed in CIRKO-TAC mice is not precipitated by coronary artery dysfunction, because CIRKO mice exhibit compensatory mechanisms (e.g., increased eNOS transcript and protein) to maintain coronary endothelial function in the setting of pressure overload.

Mitochondrial monoamine oxidase‐A‐mediated hydrogen peroxide generation enhances 5‐hydroxytryptamine‐induced contraction of rat basilar artery

We evaluated the role(s) of monoamine oxidase (MAO)-mediated H₂O₂ generation on 5-hydroxytryptamine (5-HT)-induced tension development of isolated basilar artery of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Basilar artery (endothelium-denuded) was isolated for tension measurement and Western blots. Enzymically dissociated single myocytes from basilar arteries were used for patch-clamp electrophysiological and confocal microscopic studies. Under resting tension, 5-HT elicited a concentration-dependent tension development with a greater sensitivity (with unchanged maximum tension development) in SHR compared with WKY (EC(50) : 28.4 ± 4.1 nM vs. 98.2 ± 9.4 nM). The exaggerated component of 5-HT-induced tension development in SHR was eradicated by polyethylene glycol-catalase, clorgyline and citalopram whereas exogenously applied H₂O₂ enhanced the 5-HT-elicited tension development in WKY. A greater protein expression of MAO-A was detected in basilar arteries from SHR than in those from WKY. In single myocytes and the entire basilar artery, 5-HT generated (clorgyline-sensitive) a greater amount of H₂O₂ in SHR compared with WKY. Whole-cell iberiotoxin-sensitive Ca(2+) -activated K(+) (BK(Ca) ) amplitude measured in myocytes of SHR was approximately threefold greater than that in WKY (at +60 mV: 7.61 ± 0.89 pA·pF(-1) vs. 2.61 ± 0.66 pA·pF(-1) ). In SHR myocytes, 5-HT caused a greater inhibition (clorgyline-, polyethylene glycol-catalase- and reduced glutathione-sensitive) of BK(Ca) amplitude than in those from WKY. 5-HT caused an increased generation of mitochondrial H₂O₂ via MAO-A-mediated 5-HT metabolism, which caused a greater inhibition of BK(Ca) gating in basilar artery myocytes, leading to exaggerated basilar artery tension development in SHR.

Molecular and Functional Characterization of a Novel Cardiac-Specific Human Tropomyosin Isoform

Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1alpha (also called alpha-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1kappa compared with TPM1alpha. This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

Role(s) of gravitational loading during developing period on the growth of rat soleus muscle fibers

Effects of gravitational loading or unloading on the gain of the characteristics in soleus muscle fibers were studied in rats. The tail suspension was performed in newborn rats from postnatal day 4 to month 3, and the reloading was allowed for 3 mo in some rats. Single expression of type I myosin heavy chain (MHC) was observed in approximately 82% of fibers in 3-mo-old controls, but the fibers expressing multiple MHC isoforms were noted in the unloaded rats. Although 97% of fibers in 3-mo-old controls had a single neuromuscular junction at the central region of fiber, fibers with multiple nerve endplates were seen in the unloaded group. Faster contraction speed and lower maximal tension development, even after normalization with fiber size, were observed in the unloaded pure type I MHC fibers. These parameters generally returned to the age-matched control levels after reloading. It was suggested that antigravity-related tonic activity plays an important role in the gain of single neural innervation and of slow contractile properties and phenotype in soleus muscle fibers.

Impact of Ischemia/Reperfusion Associated TnI Degradation on Cross-Bridge Dynamics in Skinned Rat Cardiac Trabeculae

Proteolytic degradation of Troponin I (cTnI) may be the cause for the depressed contractility that is seen in myocardial stunning. Here, we studied the impact of a proteolytic fragment cTnI63-193 (identified by McDonough et al, 1999) on cross-bridge cycling dynamics in isolated myocardium. Murine cTnI63-193 mutant, as well as wild type cTnC and cTnT, were expressed in E.coli. Next, FPLC purified Troponin (cTn) complex containing either wild type cTnI or cTnI63-193 was exchanged for endogenous cTn in skinned rat cardiac trabeculae; Western blot analysis confirmed that >75% cTn was exchanged. Myofilament chemo-mechanical cross-bridge dynamics were determined as function of [Ca2+] at SL=2.2 µm using an enzyme-coupled UV absorbance technique (de Tombe & Stienen, 1995).Compared to wild-type exchange, cTnI63-193 exchanged fibers displayed ∼30% decrease in myofilament Ca2+ sensitivity (EC50). In contrast, neither maximal tension development, nor maximal ATPase activity (and consequently tension-cost), were significant different between the groups, nor was cooperative thin filament activation (Hill coefficient). We conclude that cTnI63-193 found following severe ischemic/reperfusion affects cardiac function predominantly via decreased myofilament Ca2+-sensitivity. Our results may benefit rational drug development aimed to prevent ischemic/reperfusion injury in patients.

N-Terminal Truncated Cardiac TnI Extends Frank-Starling Response of the Heart

Cardiac TnI (cTnI) has an N-terminal extension containing two protein kinase A (PKA) phosphorylation sites, Ser23/24, and its removal by restrictive proteolysis in cardiac adaptation improves myocardial relaxation. Through transgenic expression of the N-terminal truncated cTnI (cTnI-ND) in the heart of endogenous cTnI knockout mice, we studied the function of cardiac muscle containing only cTnI-ND. The pure cTnI-ND hearts showed no hypertrophy or dilation with normal baseline function as compared to wild type controls (WT), confirming the non-destructive nature of cTnI-ND. Echocardiography found increased left ventricular end diastolic dimension in cTnI-ND hearts as compared to WT, implying increased relaxation and compliance in vivo. A series of preloads was tested in ex vivo working hearts for the effects of cTnI-ND on Frank-Starling response. cTnI-ND hearts showed responses to 5-10 mmHg preloads similar to that of WT. When preload was increased from 10 to 20 mmHg, cTnI-ND hearts exhibited better maintained left ventricular relaxation velocity, lower left ventricular end diastolic pressure and larger stroke volume responses than that of WT hearts. 10 nM isoproterenol further increased the positive effects of cTnI-ND on the responses of cardiac function to preloads, indicating that cTnI-ND enhances Frank-Starling relationship in the absence of direct effect of PKA phosphorylation at Ser23/24. Slack sarcomere length in isolated cardiomyocytes and the optimal sarcomere length for maximum tension development in intact left ventricular papillary muscle from cTnI-ND mice were similar to that of WT, suggesting that cTnI-ND extends the range of Frank-Starling response to high preloads by enhancing contractility independent of further increases in sarcomere length. The results demonstrated that the removal of cTnI N-terminal extension by restrictive proteolysis provides a novel posttranslational modification to utilize Frank-Starling mechanism in cardiac adaptation to physiological and pathological stresses.

Molecular and Functional Characterization of a Novel Cardiac Specific Human Tropomyosin Isoform

Previous work using human cardiac RNA identified a novel tropomyosin (TM) isoform designated as TPM1-kappa. We developed a TPM1-kappa specific antibody and quantified the levels of TPM1-kappa protein in the hearts of normal and various cardiomyopathy patients. Our study reveals that TPM1-kappa protein is expressed in the human heart and its level is differentially regulated during cardiomyopathic conditions. To investigate the role of TPM1-kappa in sarcomeric function, we generated several lines of transgenic (TG) mice overexpressing TPM1-kappa in the hearts. Immunohistochemical studies show the incorporation of the TPM1-kappa protein in the myofilaments but no significant pathological alterations are observed in these TG mice. Hearts that express TPM1-kappa protein exhibit significant decreases in rates of contraction and relaxation when assessed by ex vivo work-performing cardiac analyses. Studies on skinned fiber bundles demonstrate decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies, including circular dichroism and in vitro actin-binding assays using recombinant TM proteins demonstrate less stability and weaker actin-binding affinity of TPM1-kappa as compared to TPM1-alpha. Energetic analyses of the structural models of TPM1 kappa and alpha isoforms reveal regions which decrease the overall stability of the TPM1 kappa/kappa and kappa/alpha coiled-coil dimers. This functional analysis of TPM1-kappa provides a possible mechanistic explanation for the isoform switch that is observed in cardiomyopathy patients.