Biomedical subjects

Gopal J Babu

Publications and source records attributed to Gopal J Babu.

10 recordsLinked to original sources

PubMed2006

Effects of h1-calponin ablation on the contractile properties of bladder versus vascular smooth muscle in mice lacking SM-B myosin.

The functional significance of smooth muscle-specific h1-calponin up-regulation in the smooth muscle contractility of SM-B null mice was studied by generating double knockout mice lacking both h1-calponin and SM-B myosin. The double knockout mice appear healthy, reproduce well and do not show any smooth muscle pathology. Loss of h1-calponin in the SM-B null mice bladder resulted in increased maximal shortening velocity (V(max)) and steady-state force generation. The force dilatation pressure, which was decreased in the SM-B null mesenteric vessels, was restored to wild-type levels in the double knockout vessels. In contrast, the half-time to maximal constriction was significantly increased in the double knockout vessels similar to that of SM-B null mice and indicating decreased shortening velocity in the double knockout vessels. Biochemical analyses showed that there is a significant reduction in smooth muscle alpha-actin levels, whereas h-caldesmon levels are increased in the double knockout bladder and mesenteric vessels, suggesting that these changes may also partly contribute to the altered contractile function. Taken together, our studies suggest that up-regulation of h1-calponin in the SM-B null mice may be necessary to maintain a reduced level of cross-bridge cycling over time in the absence of SM-B myosin and play an important role in regulating the smooth muscle contraction.

Actins↗

PubMed2005

Targeted overexpression of sarcolipin in the mouse heart decreases sarcoplasmic reticulum calcium transport and cardiac contractility.

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca(2+) ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by beta-adrenergic agonists.

Animals↗

PubMed2005

Overexpression of sarcolipin decreases myocyte contractility and calcium transient.

OBJECTIVE: Sarcolipin (SLN) is a novel 31-amino-acid protein associated with the sarcoplasmic reticulum (SR) whose function in cardiac muscle is poorly defined. In this study, we tested the hypothesis that SLN is a regulator of SR Ca(2+) transport function by overexpressing SLN in adult rat ventricular myocytes which express low levels of SLN. METHODS: Expression of SLN mRNA in rat tissues was analyzed by Northern blot as well by RT-PCR analysis. To define the role of SLN in cardiac muscle contractility, we overexpressed SLN in adult rat ventricular myocytes using adenoviral gene transfer techniques. Localization of SLN in the adult rat ventricular myocytes was determined using confocal microscopy. Myocyte contractility and calcium transients were measured using edge detection and Fura 2AM. RESULTS: Our results demonstrate that overexpression of SLN decreased the cell shortening significantly when compared to control myocytes, whereas the time to peak contraction was not altered. In addition, SLN overexpression prolonged the time of 50% relaxation. Calcium transient analysis shows that time to 50% decay of [Ca(2+) ]i was markedly prolonged in SLN-overexpressing myocytes (control -245.0+/-3.78 vs. SLN -199.0+/-3.25 ms, p<0.001). However, there were no significant differences in peak amplitudes of [Ca(2+)](i) between SLN-overexpressing and control myocytes. We further demonstrate that SLN is localized within the SR membrane similar to PLB and SR Ca(2+) ATPase. Co-immunoprecipitation studies indicate that SLN can physically interact with phospholamban. CONCLUSIONS: We conclude that SLN may play an important role in regulating the SR calcium ATPase pump, possibly by interacting with phospholamban.

Adenoviridae↗

PubMed2005

Constriction velocities of renal afferent and efferent arterioles of mice are not related to SMB expression.

BACKGROUND: Constriction of renal arterioles contributes significantly to the control of perfusion and glomerular filtration. Afferent but not efferent arterioles express smooth muscle myosin heavy chain B (SMB) (with a 5'-insert of seven amino acids). The aim of the present study was to investigate (1) the constriction characteristics of afferent and efferent arterioles under physiologic load and (2) whether expression of SMB may causally contribute to these constriction characteristics. METHODS: We compared constriction parameters [constriction amplitude, maximal rate of constriction velocity ("dc/dt(max)"), and time to half-maximal constriction (t(1/2)) of in vitro perfused renal afferent and efferent arterioles of wild-type (smb(+/+)] and homozygous SMB knockout [smb(-/-)] mice upon stimulation with angiotensin II (Ang II) (10(-8) mol/L) and potassium chloride (KCl) (100 mmol/L). SMB expression was investigated by double-labeling immunofluorescence. RESULTS: Contraction amplitude and dc/dt(max) of mouse afferent arterioles upon Ang II stimulation were significantly greater compared to efferent arterioles. However, constriction amplitudes, dc/dt(max), and t(1/2) of afferent as well as efferent arterioles upon Ang II stimulation were similar in smb(+/+) and smb(-/-) mice. Constriction amplitudes upon KCl stimulation of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. Furthermore, KCl-induced dc/dt(max) and t(1/2) of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. SMB expression could be detected in afferent but not efferent arterioles in smb(+/+) mice. No SMB expression in either arteriole could be observed in smb(-/-) mice. CONCLUSION: Our results suggest that the presence of different alternatively 5'-spliced smooth muscle-myosin heavy chain (SM-MHC) isoforms does not dominate the different contractile features of physiologically loaded renal afferent or efferent arterioles.

Alternative Splicing↗

PubMed2005

Transgenic mouse models for cardiac dysfunction by a specific gene manipulation.

The sarcoplasmic reticulum Ca2+ ATPase (SERCA) plays a pivotal role in calcium cycling and the beat-to-beat function of the heart. Recent studies have shown that decreased expression and activity of SERCA are associated with end-stage heart failure in humans and in experimental animal models of heart failure. There has been considerable controversy over whether a decrease in SERCA level is a cause or effect of hypertrophy. To address directly whether alterations in SERCA levels modify calcium homeostasis and heart function, we have chosen to alter the SERCA protein expression genetically using transgenic and gene-targeted knockout mouse technology. This chapter describes the methodology for generation of mouse models that overexpress different SERCA isoforms and a SERCA2 knockout mouse model with decreased SERCA levels.

Animals↗

PubMed2004

Isoform switching from SM-B to SM-A myosin results in decreased contractility and altered expression of thin filament regulatory proteins.

We previously generated an isoform-specific gene knockout mouse in which SM-B myosin is permanently replaced by SM-A myosin. In this study, we examined the effects of SM-B myosin loss on the contractile properties of vascular smooth muscle, specifically peripheral mesenteric vessels and aorta. The absence of SM-B myosin leads to decreased velocity of shortening and increased isometric force generation in mesenteric vessels. Surprisingly, the same changes occur in aorta, which contains little or no SM-B myosin in wild-type animals. Calponin and activated mitogen-activated protein kinase expression is increased and caldesmon expression is decreased in aorta, as well as in bladder. Light chain-17b isoform (LC(17b)) expression is increased in aorta. These results suggest that the presence or absence of SM-B myosin is a critical determinant of smooth muscle contraction and that its loss leads to additional changes in thin filament regulatory proteins.

Animals↗

PubMed2004

Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins.

Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR.

Animals↗

PubMed2003

Time course of airway mechanics of the (+)insert myosin isoform knockout mouse.

Two smooth muscle myosin heavy chain isoforms that differ by the presence ([+]insert) or the absence ([-]insert) of a 7-amino acid insert in the motor domain have a 2-fold difference in their in vitro actin filament velocity. We hypothesized that a preferential expression of the fast (+)insert isoform in airway smooth muscle would increase the rate of bronchoconstriction. To verify our hypothesis we measured the time course of bronchoconstriction following a bolus injection of methacholine (160 microg/kg) in (+)insert isoform knockout (KO) and corresponding wild-type (WT) mice. Neither baseline airway resistance (Raw) (0.424 +/- 0.04 for WT and 0.374 +/- 0.01 cm H(2)O.s.ml(-1) for KO) nor peak Raw (4.1 +/- 0.9 for WT and 4.0 +/- 0.5 cm H(2)O.s.ml(-1) for KO) differed between groups. However, the time to peak Raw was significantly longer in the KO (17.2 +/- 0.6 s) compared with the WT (14.6 +/- 0.8 s) mice (P < 0.05). Differentiating Raw with respect to time revealed a greater rate of bronchoconstriction for the WT during the initial 4 s, presumably reflecting the faster shortening velocities under these relatively unloaded conditions. Reverse transcriptase-polymerase chain reaction analysis revealed that the (+)insert myosin isoform mRNA content in the WT airways was 47.8 +/- 5.6%. We conclude that the presence of the (+)insert myosin isoform in the airways increases the rate of bronchoconstriction.

Animals↗

PubMed2003

Differential vasoconstrictions induced by angiotensin II: role of AT1 and AT2 receptors in isolated C57BL/6J mouse blood vessels.

Genetically altered mice are increasingly used as experimental models. However, ANG II responses in mouse blood vessels have not been well defined. Therefore, the aim of this study was to determine the role of ANG II in regulating major blood vessels in C57/BL6J mice with isometric force measurements. Our results showed that in mouse abdominal aorta ANG II induced a concentration-dependent contraction (EC50 4.6 nM) with a maximum contraction of 75.1 +/- 4.9% at 100 nM compared with that of 60 mM K+. Similarly, femoral artery also exhibited a contractile response of 76.0 +/- 3.4% to the maximum concentration of ANG II (100 nM). In contrast, ANG II (100 nM)-induced contraction was significantly less in carotid artery (24.5 +/- 6.6%) and only minimal (3.5 +/- 0.31%) in thoracic aorta. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester and the AT2 antagonist PD-123319 failed to enhance ANG II-induced contractions. However, an AT1 antagonist, losartan (10 microM), completely inhibited ANG II (100 nM) response in abdominal aorta and carotid artery. An AT1 agonist, [Sar1]-ANG II (100 nM), behaved similarly to ANG II (100 nM) in abdominal aorta and carotid artery. RT-PCR analyses showed that mouse thoracic aorta has a significantly lower AT1 mRNA level than abdominal aorta. These results demonstrate that major mouse vessels exhibit differential contractions to ANG II, possibly because of varied AT1 receptor levels.

Angiotensin II↗

PubMed2003

The smooth muscle myosin seven amino acid heavy chain insert's kinetic role in the crossbridge cycle for mouse bladder.

The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (-/-) and a heterozygous type (+/-). For skinned muscle, an increase in MgADP or inorganic phosphate (Pi) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and Pi release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca2+ concentration in both the +/+ and +/- mouse types. However, the -/- type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the -/- type compared to the +/+ or +/- types. Exogenous Pi led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to Pi release. In addition, the increase in Pi showed similar changes in the +/+ and -/- types whereas in the +/- type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle.

Adenosine Diphosphate↗