Spontaneously Hypertensive Rats Myocytes Research Articles

Prolonged Ca2+ release refractoriness and T-tubule disruption as determinants of increased propensity to cardiac alternans in the hypertensive heart disease.

Cardiac alternans is a dynamical phenomenon linked to the genesis of severe arrhythmias and sudden cardiac death. It has been proposed that alternans is caused by alterations in Ca2+ handling by the sarcoplasmic reticulum (SR), in both the SR Ca2+ uptake and release processes. The hypertrophic myocardium is particularly prone to alternans, but the precise mechanisms underlying its increased vulnerability are not known. Mechanical alternans (intact hearts) and Ca2+ alternans (cardiac myocytes) were studied in spontaneously hypertensive rats (SHR) during the first year of age after the onset of hypertension and compared with age-matched normotensive rats. Subcellular Ca2+ alternans, T-tubule organization, SR Ca2+ uptake, and Ca2+ release refractoriness were measured. The increased susceptibility of SHR to high-frequency-induced mechanical and Ca2+ alternans appeared when the hypertrophy developed, associated with an adverse remodeling of the T-tubule network (6 mo). At the subcellular level, Ca2+ discordant alternans was also observed. From 6 mo of age, SHR myocytes showed a prolongation of Ca2+ release refractoriness without alterations in the capacity of SR Ca2+ removal, measured by the frequency-dependent acceleration of relaxation. Sensitizing SR Ca2+ release channels (RyR2) by a low dose of caffeine or by an increase in extracellular Ca2+ concentration, shortened refractoriness of SR Ca2+ release, and reduced alternans in SHR hearts. The tuning of SR Ca2+ release refractoriness is a crucial target to prevent cardiac alternans in a hypertrophic myocardium with an adverse T-tubule remodeling.

Mechanisms Of Akap150 In Aerobic Exercise-induced Improvement Of Cerebral Arterial Function In Essential Hypertension

A-kinase anchoring protein AKAP150, facilitating transduction events by binding protein kinase Cα (PKCα) to specific cellular microdomains, plays a central role in voltage-gated L-type Ca2+ channel (LTCC) remodeling in vascular smooth muscle during hypertension. Emerging evidence have demonstrated the beneficial effects of regular exercise on reducing blood pressure and improving arterial function in hypertension, however, less is known regarding the cellular mechanisms underlying the vascular changes with exercise. PURPOSE: To investigate the mechanism of AKAP150/PKCα signaling pathway in exercise-mediated LTCC function of cerebral arteries during hypertension. METHODS: 12-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were randomly assigned to sedentary (WKY-SED, SHR-SED) and exercise training (WKY-EX, SHR-EX) groups. Exercise groups were performed a moderate-intensity treadmill running (about 55-65% VO2max, 20 m/min, 0 grade, 60 min, 5 days/week). After 12 weeks, artery contraction myography, patch-clamp electrophysiology, Ca2+ image, Western blot and immunofluorescence were used to detect cerebral vascular tone, LTCC whole-cell and single channel currents, Ca2+ sparklets, and AKAP150/PKCα signaling pathway. RESULTS: Exercise attenuated LTCC contribution to cerebral vascular tone regulation (39.0±1.3 vs. 27.5±2.0 %Kmax) and LTCC currents in cerebral arterial myocytes of SHR (−13.2±1.7 vs. −9.6±1.5 pA/pF, both P<0.05). The LTCC channel open probability (nPo) and persistent Ca2+ sparklet activity (nPs) of cerebral arterial myocytes were significantly reduced in SHR-EX as compared with SHR-SED (nPo: 0.08±0.01 vs. 0.12±0.01; nPs: 0.65±0.15 vs. 0.82±0.10; both P<0.05). The protein expression of AKAP150 in cerebral artery was significantly up-regulated in SHR-SED (4.9±0.6), while down-regulated in SHR-EX (1.4±0.2, P<0.05). The colocalization rate of AKAP150 and PKCα at the sarcolemma were lower in cerebral arterial myocytes from SHR-EX than those from SHR-SED (20.1±1.1 vs. 30.0±1.6%, P<0.05). CONCLUSIONS: Chronic exercise inhibits LTCC channel activity and persistent Ca2+ sparklets in vascular smooth muscle via suppression of AKAP150/PKCα signaling pathway, and ameliorates the dysfunction of cerebral arteries during hypertension.

Change in late sodium current of atrial myocytes in spontaneously hypertensive rats with allocryptopine treatment.

We aimed to study the effect of allocryptopine (All) on the late sodium current (INa,Late) of atrial myocytes in spontaneously hypertensive rats (SHR). The enzyme digestion method was used to separate single atrial myocytes from SHR and Wistar-Kyoto (WKY) rats. INa,Late was recorded using the patch-clamp technique, and the effect of All was evaluated on the current. Compared with WKY rat cells, an increase in the INa,Late current in SHR myocytes was found. After treatment with 30 µM All, the current densities were markedly decreased; the ratio of INa,Late/INa,peak of SHR was reduced by 30 µM All. All reduced INa,Late by alleviating inactivation of the channel and increasing the window current of the sodium channel. Furthermore, INa,Late densities of three SCN5A mutations declined substantially with 30 µM All in a concentration-dependent manner. The results clearly show that an increase in INa,Late in SHR atrial myocytes was inhibited by All derived from Chinese herbal medicine.

Increased Susceptibility of Spontaneously Hypertensive Rats to Ventricular Tachyarrhythmias during the Early Stages of Hypertension

Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation (VT/VF). We hypothesized that during the early stages of uncontrolled hypertension, the susceptibility to otherwise benign stress promotes VT/VF. To test this hypothesis we compared the susceptibility of 5-6-month-old male spontaneously hypertensive rats (SHR) and age/gender-matched normotensive rats (NR) to VT/VF during challenge with mild oxidative stress (H2O2, 0.15 mmol/L). H2O2 initiated spontaneous VT in all 30 SHR but none of the NR hearts. In 33% of SHR cases, focal VT degenerated to VF within 3 s. Simultaneous voltage-Ca optical mapping of Langendorff-perfused SHR hearts revealed that H2O2-induced VT/VF arose spontaneously from focal activations at the base and mid left ventricular epicardium. Microelectrode recording of SHR hearts showed that VT was initiated by early afterdepolarization (EAD)-mediated triggered activity. However, despite the increased susceptibility of SHR hearts to VT/VF, patch-clamped isolated SHR ventricular myocytes developed EADs and triggered activity to the same extent as NR ventricular myocytes, except that SHR myocytes had a relatively larger EAD amplitude. Histopathological and echocardiographic studies showed that compared to NR, SHR hearts exhibited increased left ventricular fibrosis and mild hypertrophy. We conclude that during the early stages of hypertension (i.e., prior to the development of overt heart failure) SHR hearts manifest increased susceptibility to VT/VF when challenged with mild oxidative stress that appears to be related to tissue remodeling (i.e., hypertrophy, fibrosis) moreso than cellular electrophysiological remodeling. Our findings highlight the need for aggressive treatment during the stages of early hypertension to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias.

Effect of fosinopril on the transient outward potassium current of hypertrophied left ventricular myocardium in the spontaneously hypertensive rat

To investigate fosinopril's effect on the transient outward potassium current (Ito) of differing degrees of hypertrophied myocytes in the spontaneously hypertensive rat (SHR). Ten- and 24-week-old SHRs were used as models for cardiac hypertrophy. Hypertrophied ventricular myocytes were exposed to 1, 10, and 100 μmol/L fosinopril; the whole-cell patch-clamp technique was used to study the effects on the transient outward potassium current. Ito current density was decreased in SHR myocytes relative to controls (14.17 ± 0.31 and 11.62 ± 0.08 pA/pF in 10- and 24-week-old SHR versus 16.73 ± 0.15 pA/pF, p < 0.01). Higher concentrations of fosinopril (10 and 100 μmol/L) increased Ito peak current density in 10-week-old SHR myocytes compared with controls (14.92 ± 0.14 and 15.27 ± 0.13 pA/pF versus 14.17 ± 0.31 pA/pF, p < 0.01). Fosinopril increased Ito peak current density in 24-week-old SHR myocytes at all doses (12.70 ± 0.07, 13.74 ± 0.10, and 14.53 ± 0.13 versus 11.62 ± 0.08 pA/pF for controls, p < 0.01). Fosinopril had a greater Ito elevation potential on hypertrophied myocytes in 24-week-old compared with 10-week-old SHR for each dose (1.08 ± 0.09 versus 0.37 ± 0.26 pA/pF, p < 0.01; 2.13 ± 0.05 versus 0.75 ± 0.35 pA/pF, p < 0.01; 2.92 ± 0.07 versus 1.10 ± 0.40 pA/pF, p < 0.01). Fosinopril increased Ito current density in hypertrophied myocytes. This effect was more pronounced in myocytes with a greater degree of hypertrophy.

Atrial endothelin-1 signalling in hypertensive rats

Purpose: Hypertension induces cardiac remodelling at the structural and functional level. Endothelin-1 (ET-1) may contribute to cardiac remodelling. It is not known, however, whether atrial ET-1 signalling is altered in hypertension. Here we tested the hypothesis that ET-1 signalling is augmented in the atria of spontaneously hypertensive rats (SHR) and that this may contribute to hypertension-induced atrial arrhythmias. Methods: SHR and control Wistar-Kyoto (WKY) rats were studied at 6-8 months of age. Hearts were removed for morphometric analysis. Atria were snap frozen and analyzed for mRNA and protein expression by qPCR and Western blotting. Isolated atrial myocytes were field stimulated in the presence of 50 nM ET-1, and Ca transients (CaTs) were characterized at the global or subcellular level by epifluorescence (Fura-2/AM) and line scan confocal microscopy (Fluo-4/AM). To mimic atrial tachycardia, atrial tissue slices were stimulated at 5 Hz for up to 20 h and used for Western blotting. Results: At 6-8 months of age, SHR exhibited compensated LV hypertrophy, but left atrial hypotrophy, despite increased atrial mRNA levels of BNP (2-fold) and β-MHC (3.6-fold), while α-MHC was unaffected. There were no differences in global CaTs between atrial myocytes from WKY and SHR. Exposure of atrial myocytes to 50 nM ET-1 caused a 35% increase in systolic Ca in SHR (n=14), but only a 16% increase in WKY (n=21; P<0.05). Moreover, in SHR atrial myocytes, ET-1 induced a larger fractional SR Ca release (P<0.05 vs WKY). The ET-1-induced increase in CaTs occurred in the subsarcolemmal as well as in the central cytoplasm. Notably, ET-1 also induced arrhythmogenic Ca waves both in SHR and WKY atrial myocytes. Further analysis at the molecular level indicated that the atrial mRNA expression of ET-1 was increased (2-fold) in SHR. ETA receptor expression was unaltered at the mRNA level but upregulated (4-fold) at the protein level in the left atria. Gαq and PLCβ (isoforms 1 and 4) mRNA expression were also upregulated in SHR. Simulated atrial tachycardia in atrial tissue slices caused a 2-fold upregulation of ETA receptor expression in both SHR and WKY. Conclusions: At 6-8 months, SHR exhibit increased atrial expression of ET-1, ETA receptors, Gαq and PLCβ. Atrial tachycardia further increases ETA receptor expression. At the functional level, atrial myocytes from SHR respond to ET-1 with increases in systolic Ca and the development of arrhythmogenic Ca waves. Thus, atrial ET-1 signalling is enhanced already at early stages of hypertension and may contribute to the initiation of atrial tachycardia in hypertension.

An Elevation in Physical Coupling of Type 1 Inositol 1,4,5-Trisphosphate (IP 3 ) Receptors to Transient Receptor Potential 3 (TRPC3) Channels Constricts Mesenteric Arteries in Genetic Hypertension

Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C–coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP 3 ) that activates sarcoplasmic reticulum IP 3 receptors. In cerebral artery myocytes, IP 3 receptors release sarcoplasmic reticulum Ca 2+ and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP 3 receptors control vascular contractility in systemic arteries and IP 3 R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ≈2.7- and 2-fold higher in mesenteric arteries of spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP 3 R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-SHRs and WKY rats. Control, IP 3 -induced and endothelin-1 (ET-1)-induced fluorescence resonance energy transfer between IP3R1 and TRPC3 was higher in SHR than WKY myocytes. IP3-induced cation current was ≈3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and calmodulin and IP 3 receptor binding domain peptide, an IP 3 R-TRP physical coupling inhibitor, reduced IP 3 -induced cation current and ET-1–induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a sarcoplasmic reticulum Ca 2+ -ATPase blocker, did not alter ET-1–stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP 3 R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP 3 R1 to TRPC3 channel molecular coupling augments ET-1–induced vasoconstriction during hypertension.

Non-steady-state calcium handling in failing hearts from the spontaneously hypertensive rat

It is generally agreed that changes in Ca(2+) cycling are often associated with heart failure, yet the impact of these changes on a beat-to-beat basis remains unclear. Measurements of isometric force and [Ca(2+)](i) were made at 37°C in left ventricular trabeculae from failing spontaneously hypertensive rat (SHR) hearts, and their normotensive Wistar-Kyoto (WKY) controls. At 1 Hz, peak stress was reduced in SHR (14.5 ± 2.4 mN mm(-2) versus 22.5 ± 6.7 mN mm⁻² for WKY), although the Ca(2+) transients were bigger (peak [Ca(2+)](i) 0.60 ± 0.08 μM versus 0.38 ± 0.03 μM for WKY) with a slower decay of fluorescence (time constant 0.105 ± 0.005 s versus 0.093 ± 0.002 s for WKY). To probe dynamic Ca(2+) cycling, two experimental protocols were used to potentiate force: (1) an interval of 30 s rest, and (2) a 30-s train of paired-pulses, and the recirculation fraction (RF) calculated for recovery to steady-state. No difference was found between rat strains for RF calculated from either peak force or Ca(2+), although the RF was dependent on potentiation protocol. Since SR uptake is slower in SHR, the lack of change in RF must be due to a parallel decrease in trans-sarcolemmal Ca(2+) extrusion. This view was supported by a slower decay of caffeine-induced Ca(2+) transients in SHR trabeculae. Confocal analysis of LV free wall showed t-tubules were distorted in SHR myocytes, with reduced intensity of NCX and SERCA2a labelling in comparison to WKY.

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.

Development of Calcium Handling Defects During Aging in Spontaneously Hypertensive Rats

Several defects in calcium handling have been identified in failing myocytes. We investigated the progression of these defects in the course of development of overt heart failure (HF). Intracellular Ca2+ transients were measured using confocal microscopy in intact hearts from age-matched Wistar-Kyoto (WKY) control rats and Spontaneously Hypertensive Rats (SHR) at 6, 7.5, 9 and >22 months of age. Amplitude of basal Ca2+ transients (cycle length of 700msec) in SHR increased at 7.5 and 9 months, but subsequently decreased at 22 months compared to WKY. SHR myocytes exhibited longer transient duration starting at 9 months compared to WKY. Cell-to-cell variability in transient duration increased at 7.5 months and subsequently decreased at 9 months as defects became more extensive. At 22 months, Ca2+ transients showed further increases in transient duration and intercellular variability. Restitution of Ca2+ release was slowed and was paralleled by increased Ca2+ alternans susceptibility starting at 9 months in SHR. Dyssynchronous alternans incidence increased beginning at 7.5 months in SHR compared to WKY. SHR myocytes also demonstrated an increased incidence of spontaneous Ca2+ waves at all ages, with the greatest difference at 22 months. A separate population of SHR myocytes showed Ca2+ waves that were activated during pacing (“triggered waves”) whose incidence increased at all ages but most profoundly at 22 months. We conclude that well-coupled failing myocytes demonstrate progressively increasing defects in Ca2+ cycling. Biphasic changes in calcium transient magnitude may partially account for a transient inotropic effect during compensation but ultimately reduced cardiac output in HF. The progression of Ca2+ handling defects may also account for the increasing sensitivity to alternans as well as incidence of spontaneous and triggered Ca2+ waves with age, possibly explaining increased arrhythmias during progressive HF.

Reduced cardiotropic response to insulin in spontaneously hypertensive rats: role of peroxisome proliferator-activated receptor-γ-initiated signaling

Vascular insulin resistance plays a crucial pathogenic role in the development of genetic hypertension. However, it is not known whether hypertension-associated myocardial insulin resistance also exists, and whether this is involved in the development of related diseases, such as hypertensive heart failure. The present study aimed to determine whether hypertension-associated myocardial insulin resistance exists, in addition to any underlying mechanism. Ventricular myocytes were enzymatically isolated from male Wistar-Kyoto (WKY) rats or spontaneously hypertensive rats (SHR) and myocyte shortening and intracellular Ca2+ transient were assessed, as was any signaling mechanism. Compared with WKY rats, insulin-stimulated myocardial glucose uptake was blunted in SHR. More importantly, the positive inotropic effect of insulin was significantly reduced in SHR myocytes. Moreover, peroxisome proliferator-activated receptor (PPAR)gamma and phosphatidylinositol 3 (PI-3) kinase p85 expression and insulin-induced Akt phosphorylation were reduced in SHR cardiomyocytes, but were markedly restored when animals were treated with rosiglitazone for 14 days. Pretreatment with an Akt inhibitor abolished the inotropic effect induced by insulin in rosiglitazone-treated SHR cardiomyocytes. The data obtained demonstrate that insulin resistance exists in SHR cardiomyocytes as manifested by both reduced insulin-stimulated glucose uptake and an impaired contractile response to insulin, which is attributable to decreased PPARgamma expression and subsequent impairment in PI-3 kinase/Akt signaling.

Cellular distribution of calcium current is unaltered during compensated hypertrophy in the spontaneously hypertensive rat

Changes in cellular calcium (Ca(2+)) handling are thought to underlie the altered contraction that occurs during cardiac hypertrophy and failure. Recent work has highlighted the importance of t-tubules in the control of intracellular Ca(2+). The present study was performed to investigate whether changes in the distribution of I (Ca) between the surface and t-tubule membranes might contribute to the altered Ca(2+) handling observed during compensated hypertrophy in the spontaneously hypertensive rat (SHR). Experiments were performed on ventricular myocytes isolated from 5-month-old SHR and normotensive Wistar-Kyoto (WKY) control rats. Osmotic shock using formamide was used to disrupt the t-tubular system and the whole-cell patch clamp technique used to monitor I (Ca) in the presence and absence of t-tubules. Membrane capacitance and I (Ca) were greater in control SHR than WKY myocytes; following detubulation, cell capacitance and I (Ca) both decreased and were no longer significantly different in the two cell types. The density of I (Ca) was not significantly different in control SHR and WKY cells or in detubulated myocytes from the two species. These data suggest that the distribution of I (Ca) is unchanged in SHR myocytes compared to WKY controls; I (Ca) density in the t-tubules was 1.2-fold greater than in the sarcolemma in both strains. These data also imply that the increase in surface area in SHR myocytes is due principally to an increase in t-tubular area, which is accompanied by an approximately equivalent increase in I (Ca), so that the density of I (Ca) at the cell surface and in the t-tubules remains the same. These changes would be expected to retain cell function and synchronicity of Ca(2+) release in the SHR at this stage of compensated hypertrophy.

Molecular and electrophysiological characteristics of K+ conductance sensitive to acidic pH in aortic smooth muscle cells of WKY and SHR

Changes in K(+) conductances and their contribution to membrane depolarization in the setting of an acidic pH environment have been studied in myocytes from aortic smooth muscle cells of spontaneously hypertensive rats (SHR) compared with those from Wistar-Kyoto (WKY) rats. The resting membrane potential (RMP) of aortic smooth muscle at extracellular pH (pH(o)) of 7.4 was significantly more depolarized in SHR than in WKY rats. Acidification to pH(o) 6.5 made this difference in RMP between SHR and WKY rats more significant by further depolarizing the SHR myocytes. Large-conductance Ca(2+)-activated K(+) (BK) currents, which were markedly suppressed by acidification, were larger in aortic myocytes of SHR than in those of WKY rats. In contrast, acid-sensitive, non-BK currents were smaller in SHR. Western blot analyses showed that expression of BK-alpha- and -beta(1) subunits in SHR aortas was upregulated and comparable with those in WKY rats, respectively. Additional electrophysiological and molecular studies showed that pH- and halothane-sensitive two-pore domain weakly inward rectifying K(+) channel (TWIK)-like acid-sensitive K(+) (TASK) channel subtypes were functionally expressed in aortas, and TASK1 expression was significantly higher in WKY than in SHR. Although the background current through TASK channels at normal pH(o) (7.4) was small and may not contribute significantly to the regulation of RMP, TASK channel activation by halothane or alkalization (pH(o) 8.0) induced significant hyperpolarization in WKY but not in SHR. In conclusion, the larger depolarization and subsequent abnormal contractions after acidification in aortic myocytes in the setting of SHR hypertension are mainly attributable to the larger contribution of BK current to the total membrane conductance than in WKY aortas.

Inhibition of Sarco(endo)plasmic Reticulum Ca&lt;SUP&gt;2+&lt;/SUP&gt;-ATPase Differentially Regulates Contractile Function in Cardiac Myocytes From Normotensive and Spontaneously Hypertensive Rats: Role of Ca&lt;SUP&gt;2+&lt;/SUP&gt; Regulatory Proteins

Hypertension leads to impaired contractile function. This study examined the impact of inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by thapsigargin or cyclopiazonic acid (CPA) on cardiac contractile function in ventricular myocytes from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Mechanical properties were examined including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ transients were evaluated as fura-2 fluorescent intensity (FFI), excitation-induced change in FFI (DeltaFFI = peak-basal), and fluorescence decay rate (tau). Expression of Ca2+ regulatory proteins SERCA2a, Na+-Ca2+ exchanger (NCX), and phospholamban (PLB) were assessed by reverse transcriptase polymerase chain reaction and Western blot. SHR rats exhibited elevated blood pressure. SHR myocytes displayed decreased PS +/- dL/dt, peak FFI, and DeltaFFI; shortened TPS; prolonged tau with normal TR90; and basal FFI compared with WKY myocytes. Inhibition of SERCA with thapsigargin (5 microM) or CPA (10 microM) significantly depressed PS +/- dL/dt, baseline FFI, and DeltaFFI, and prolonged TPS, TR90, and tau in WKY myocytes. However, SHR myocytes were relatively insensitive to thapsigargin or CPA with only TPS and TR90 prolonged. Both mRNA and protein expressions of NCX and PLB were significantly enhanced, whereas SERCA2a protein abundance was reduced in SHR rats compared with the WKY group. Our data suggest that inhibition of SERCA function differentially affected cardiac contractile function in ventricular myocytes from normotensive and hypertensive rats possibly through reduced SERCA2a, elevated PLB, and NCX expression under hypertension.

Decreased Ca2+extrusion via Na+/Ca2+exchange in epicardial left ventricular myocytes during compensated hypertrophy

Hypertension-induced cardiac hypertrophy alters the amplitude and time course of the systolic Ca2+ transient of subepicardial and subendocardial ventricular myocytes. The present study was designed to elucidate the mechanisms underlying these changes. Myocytes were isolated from the left ventricular subepicardium and subendocardium of 20-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY; control). We monitored intracellular Ca2+ using fluo 3 or fura 2; caffeine (20 mmol/l) was used to release Ca2+ from the sarcoplasmic reticulum (SR), and Ni2+ (10 mM) was used to inhibit Na+/Ca2+ exchange (NCX) function. SHR myocytes were significantly larger than those from WKY hearts, consistent with cellular hypertrophy. Subepicardial myocytes from SHR hearts showed larger Ca2+ transient amplitude and SR Ca2+ content and less Ca2+ extrusion via NCX compared with subepicardial WKY myocytes. These parameters did not change in subendocardial myocytes. The time course of decline of the Ca2+ transient was the same in all groups of cells, but its time to peak was shorter in subepicardial cells than in subendocardial cells in WKY and SHR and was slightly prolonged in subendocardial SHR cells compared with WKY subendocardial myocytes. It is concluded that the major change in Ca2+ cycling during compensated hypertrophy in SHR is a decrease in NCX activity in subepicardial cells; this increases SR Ca2+ content and hence Ca2+ transient amplitude, thus helping to maintain the strength of contraction in the face of an increased afterload.

Transmural changes in size, contractile and electrical properties of SHR left ventricular myocytes during compensated hypertrophy.

Some hypertrophic stimuli provoke responses from myocytes that vary across the thickness of the left ventricular wall. The Spontaneously Hypertensive Rat (SHR) is a well-established genetic model of hypertension and whole heart hypertrophy. Details of transmural responses to hypertension in the SHR are few, but are needed if the properties of this model are to be fully understood. We therefore tested the hypothesis that left ventricular myocytes of the SHR do not respond uniformly to their hypertensive environment. The volume, contraction and action potentials of enzymically isolated sub-epicardial (EPI) mid-myocardial (MID) and sub-endocardial (ENDO) myocytes from the left ventricle of 20-week-old SHR and normotensive Wistar-Kyoto (WKY) control rats were compared. Compared to WKY, as a single population, SHR myocytes displayed concentric hypertrophy (larger volumes with smaller length:width) increased t-tubule spacing, larger and prolonged cell shortening and intracellular calcium ([Ca2+]i) transients and longer action potentials. However, these responses differed across the left ventricular wall. MID myocytes showed significantly less hypertrophy than EPI and ENDO myocytes. EPI myocytes showed the largest (and significant) increases in cell shortening, [Ca2+]i transients and action potential duration, whilst MID myocytes showed the smallest (and non-significant) changes in these parameters. Real time reverse transcription polymerase chain reaction analysis on cardiac tissue suggest that increased expression of mRNA for fibronectin-1 and protein kinase Cepsilon are involved in the hypertrophic response of the whole heart. Our findings show that in the SHR, the effect of hypertension upon the morphology, mechanical activity and electrical activity of left ventricular myocytes is dependent upon their transmural location. Therefore, in addition to the overall compensating response to hypertension, i.e. increased contractility, there are likely to be regionally specific alterations in mechano-electric interactions that may influence the properties of this important model, e.g. its pre-disposition to arrhythmia whilst still in a compensated state.

Downregulation of the BK channel beta1 subunit in genetic hypertension.

The molecular mechanisms underlying increased arterial tone during hypertension are unclear. In vascular smooth muscle, localized Ca2+ release events through ryanodine-sensitive channels located in the sarcoplasmic reticulum (Ca2+ sparks) activate large-conductance, Ca2+-sensitive K+ (BK) channels. Ca2+ sparks and BK channels provide a negative feedback mechanism that hyperpolarizes smooth muscle and thereby opposes vasoconstriction. In this study, we examined Ca2+ sparks and BK channel function in Wistar-Kyoto (WKY) rats with borderline hypertension and in spontaneously hypertensive rats (SHR), a widely used genetic model of severe hypertension. We found that the amplitude of spontaneous BK currents in WKY and SHR cells were smaller than in normotensive cells even though Ca2+ sparks were of similar magnitude. BK channels in WKY and SHR cells were less sensitive to physiological changes in intracellular Ca2+ than normotensive cells. Our data indicate that decreased expression of the BK channel beta1 subunit underlies the lower Ca2+ sensitivity of BK channels in SHR and WKY myocytes. We conclude that the lower expression of the beta1 subunit during genetic borderline and severe hypertension reduced BK channel activity by decreasing the sensitivity of these channels to physiological changes in Ca2+. These results support the view that changes in the molecular composition of BK channels may be a fundamental event contributing to the development of vascular dysfunction during hypertension.

Influence of hypertension on cardiac contractile response of human erythrocyte-derived depressing factor in ventricular myocytes.

Erythrocyte-derived depressing factor (EDDF), a novel hypotensive factor purified from human erythrocytes, elicits endothelium-dependent vasorelaxation by reducing intracellular Ca2+ in vascular smooth muscle cells. However, its cardiac response is unknown. This study was designed to examine the cardiac contractile response of EDDF under both normotensive and hypertensive conditions. Ventricular myocytes were isolated from adult male spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) normotensive rats. Mechanical properties were evaluated using an IonOptix MyoCam system and intracellular Ca2+ was measured with fura-2 fluorescence. Myocytes were electrically stimulated to contract at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% re-lengthening (TR(90)), maximal velocity of shortening/re-lengthening (+/- dl/dt), fura-fluorescence intensity change (DeltaFFI), and fura-fluorescence decay rate (tau). SHR rats displayed significantly elevated blood pressure. EDDF (10-9-10-4 g/ml) did not affect PS, TPS, TR(90), DeltaFFI and tau but depressed +/- dl/dt at higher doses in WKY myocytes. However, EDDF depressed PS, +/- dl/dt and DeltaFFI, shortened TPS without affecting TR(90) and tau in SHR myocytes. Pretreatment of the myocytes with the nitric oxide synthase inhibitor Nvarpi-nitro-l-arginine methyl ester (l-NAME) did not affect the EDDF-induced inhibition of PS and +/- dl/dt in SHR myocytes but unmasked an EDDF-induced negative response in WKY myocytes. These data indicate that EDDF may participate in the modulation of cardiac contractile function under hypertensive, but not normotensive, conditions. The cardiac depressive effect of EDDF is unlikely due to release of nitric oxide, as suggested in vascular smooth muscles.

Ramipril treatment alters Ca 2+ and K + channels in small mesenteric arteries from Wistar-Kyoto and spontaneously hypertensive rats

Numerous studies have emphasized the important role of altered Ca 2+ channel function in hypertension. We previously showed that Ca 2+ currents measured in myocytes isolated from both Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) small mesenteric arteries closely correlated with systolic blood pressure (BP) during normal development. The purpose of the present experiments was to determine whether antihypertensive therapy with an angiotensin converting enzyme inhibitor normalizes Ca 2+ channel function in SHR myocytes along with BP. Ramipril (3.5 mg/kg/day) was added to the drinking water of 12-week-old male WKY and SHR for 8 weeks. Segments of small mesenteric arteries were used for isometric contraction studies, and for isolation of myocytes for measurement of Ca 2+ and K + currents (I Ca and I K) by patch clamp methods. Ramipril treatment decreased systolic pressure in WKY and SHR, decreased heart weight and heart weight-to-body weight ratio in SHR, and decreased body weight in WKY. Maximum contractile responses to Bay k 8644 in SMA from ramipril-treated SHR were smaller compared to untreated SHR (10% ± 2% v 55% ± 7% of the response to 120 mmol/L KCl). The smaller responses in WKY were not affected by ramipril treatment (11% ± 4% v 8% ± 3%). Contractile responses to 10 mmol/L tetraethylammonium (TEA) were not different in untreated versus ramipril-treated SHR (65% ± 6% v 82% ± 8%) but were increased in treated WKY (4% ± 1% v 35% ± 9%). Ramipril treatment decreased peak I Ca and equalized the voltage-dependence of I Ca activation between SHR and WKY. The I K measured from holding potentials of −60 and −20 mV were significantly smaller in treated SHR and WKY compared to their untreated counterparts, as was the component of I K measured in the presence of 100 nmol/L iberiotoxin. These results show that ramipril treatment decreases arterial pressure and Ca 2+ channel function in SHR as expected but unexpectedly also decreases I K in both WKY and SHR. These results suggest that angiotensin may have a BP independent effect on ion channel function in arterial smooth muscle.

Differences in K + current components in mesenteric artery myocytes from WKY and SHR

Previous studies have documented increased K + permeability of arterial smooth muscle in hypertension and suggested a role in altered arterial contractile function. To characterize the mechanisms responsible for these alterations, we determined the contribution of K + current (I K) components to whole cell I K in freshly dispersed myocytes and tetraethylammonium (TEA)-induced contractile responses in mesenteric arteries of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Tetraethylammonium produced a larger tonic contractile response in SHR with a lower threshold compared to WKY (ie, 0.1 v 1 mmol/L), which was due in part to the larger Ca 2+ current in SHR. Whole cell I K recorded by perforated patch methods was similar at a holding potential (HP) of −60 mV (I K60), but were larger in SHR when recorded from a HP of −20 mV (I K20). The selective blocker iberiotoxin (IbTX) was used to separate the contribution of voltage- (K V) and calcium-dependent (K Ca) components of I K60. The I K60 and I K20 component inhibited by 100 nmol/L IbTX (ie, K Ca) was larger in SHR than in WKY myocytes, whereas the IbTX-insensitive I K60 component (ie, K V) was larger in WKY. In the presence of IbTX, 1 and 10 mmol/L TEA inhibited a larger fraction of I K60 in SHR myocytes compared with WKY. The activation properties of the TEA-sensitive and TEA-insensitive K V components determined by fitting a Boltzmann activation function to the current-voltage data, exhibited both group and treatment differences in the half maximal activation voltage (V 0.5). The V 0.5 of the TEA-sensitive K V component was more positive than that of the TEA-insensitive component in both groups, and values for the V 0.5 of both TEA-sensitive and TEA-insensitive components were more negative in SHR than WKY. These results show that SHR myocytes have larger K Ca and smaller K V current components compared with WKY. Furthermore, SHR myocytes have a larger TEA-sensitive K V component. These differences may contribute to the differences in TEA contractions, resting membrane potential, Ca 2+ influx, and K Ca current reported in hypertensive arteries.