Exercise training attenuates coronary smooth muscle phenotypic modulation and nuclear Ca2+ signaling

Physical inactivity is an independent risk factor for coronary heart disease, yet the mechanism(s) of exercise-related cardioprotection remains unknown. We tested the hypothesis that coronary smooth muscle after exercise training would have decreased mitogen-induced phenotypic modulation and enhanced regulation of nuclear Ca(2+). Yucatan swine were endurance exercise trained (EX) on a treadmill for 16-20 wk. EX reduced endothelin-1-induced DNA content by 40% compared with sedentary (SED) swine (P < 0.01). EX decreased single cell peak endothelin-1-induced cytosolic Ca(2+) responses compared with SED by 16% and peak nuclear Ca(2+) responses by 33% (P < 0.05), as determined by confocal microscopy. On the basis of these results, we hypothesized that sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and intracellular Ca(2+) stores in native smooth muscle are spatially localized to dissociate cytosolic Ca(2+) and nuclear Ca(2+). Subcellular localization of SERCA in living and fixed cells revealed a distribution of SERCA near the sarcolemma and on the nuclear envelope. These results show that EX enhances nuclear Ca(2+) regulation, possibly via SERCA, which may be one mechanism by which coronary smooth muscle cells from EX are less responsive to mitogen-induced phenotypic modulation.     

Chronic and acute exercise do not alter Ca2+ regulatory systems and ectonucleotidase activities in rat heart.

The purpose of this investigation was to examine the effects of chronic and acute exercise on the main components involved in excitation-contraction coupling and relaxation in rat heart. Sixty male Wistar rats were divided into a sedentary (S) and three 12-wk treadmill-trained groups (T-1, moderate intensity; T-2, high intensity; T-3, interval running). After 12-wk, 15 rats from the S group and 15 rats from the T-2 group were subjected to a single treadmill-exercise session until exhaustion before being killed at 0, 24, or 48 h (acute exercise). The remaining animals were killed 48 h after the last standard exercise session (chronic exercise). The efficacy of the training programs was confirmed by an increase in treadmill endurance time and in skeletal muscle citrate synthase activity. None of the exercise programs modified heart weight or cardiac oxidative capacity. [(3)H]PN200-110 and [(3)H]ryanodine binding to cardiac homogenates indicated that the density of L-type and sarcoplasmic reticulum (SR) Ca(2+) channels was the same in S and trained rats. The SR Ca(2+)-ATPase activity was also unmodified. Finally, the activities of the ectoenzymes Mg(2+)-ATPase and 5'-nucleotidase, which are involved in degradation of extracellular nucleotides, were not affected by either of the running programs. After the acute exercise session, no changes were detected in either of the tested parameters in heart homogenates of S and T-2 animals. We conclude that neither treadmill-exercise training for 12 wk nor exhaustive exercise alters the density of Ca(2+) channels involved in excitation-contraction coupling or the SR Ca(2+)-ATPase and the ectonucleotidase activities in rat heart.     

Effect of exercise training on intracellular free Ca2+ transients in ventricular myocytes of rats.

The purpose of this study was to test the hypothesis that exercise training induces enhanced intracellular free Ca2+ (Cai) availability to the contractile elements of cardiac cells. Cai transients were directly measured in single isolated contracting ventricular myocytes from exercise-trained (EX) and sedentary control (SED) rats. Male Sprague-Dawley rats underwent 16 wk of progressive treadmill exercise (32 m/min, 8% grade, 1.5 h/day) (EX) or were cage confined (SED). EX rats had lower resting heart rate and elevated skeletal muscle oxidative capacity. Cai was measured with the fluorescent Cai indicator fura-2. Simultaneous video monitoring indicated that myocytes suspended in physiological salt solution were quiescent until stimulated electrically at a frequency of 0.2 Hz (12-36 V, 2-ms duration). Stimulated Cai transients, measured from changes in fura-2 fluorescence, were similar in cells from EX and SED groups. Peak shortening, time to peak shortening, velocity of shortening, contraction duration, and time to half-relaxation were also similar in cells from EX and SED rats. Ryanodine (10 microM) was applied to eliminate the contribution of Ca2+ release from sarcoplasmic reticulum to the Cai transient. Verapamil was applied to eliminate the contribution of voltage-gated Ca2+ channels to Cai transients. Cai transients were also similar in cells from EX and SED groups after these pharmacological interventions. These results suggest that treadmill training of rats does not alter Cai availability to the contractile elements in isolated ventricular myocytes.     

Overexpression of type 1 angiotensin II receptors impairs excitation-contraction coupling in the mouse heart

Transgenic mice that overexpress human type 1 angiotensin II receptor (AT(1)R) in the heart develop cardiac hypertrophy. Previously, we have shown that in 6-mo AT(1)R mice, which exhibit significant cardiac remodeling, fractional shortening is decreased. However, it is not clear whether altered contractility is attributable to AT(1)R overexpression or is secondary to cardiac hypertrophy/remodeling. Thus the present study characterized the effects of AT(1)R overexpression on ventricular L-type Ca(2+) currents (I(CaL)), cell shortening, and Ca(2+) handling in 50-day and 6-mo-old male AT(1)R mice. Echocardiography showed there was no evidence of cardiac hypertrophy in 50-day AT(1)R mice but that fractional shortening was decreased. Cellular experiments showed that cell shortening, I(CaL), and Ca(v)1.2 mRNA expression were significantly reduced in 50-day and 6-mo-old AT(1)R mice compared with controls. In addition, Ca(2+) transients and caffeine-induced Ca(2+) transients were reduced whereas the time to 90% Ca(2+) transient decay was prolonged in both age groups of AT(1)R mice. Western blot analysis revealed that sarcoplasmic reticulum Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger protein expression was significantly decreased in 50-day and 6-mo AT(1)R mice. Overall, the data show that cardiac contractility and the mechanisms that underlie excitation-contraction coupling are altered in AT(1)R mice. Furthermore, since the alterations in contractility occur before the development of cardiac hypertrophy, it is likely that these changes are attributable to the increased activity of the renin-angiotensin system brought about by AT(1)R overexpression. Thus it is possible that AT(1)R blockade may help maintain cardiac contractility in individuals with heart disease.     

Exercise training increases the Ca(2+) sensitivity of tension in rat cardiac myocytes.

The heart is known to respond to a program of chronic exercise in ways that enhance cardiac function. However, the cellular mechanisms involved in training-induced improvements in the contractile function of the myocardium are not known. In this study we tested the hypothesis that increased contractility of the myocardium associated with exercise training is due, in part, to increases in the Ca(2+) sensitivity of steady-state tension. Female Sprague-Dawley rats were randomly divided into sedentary control (C) and exercise-trained (T) groups. The T rats underwent 11 wk of progressive treadmill exercise (1 h/day, 5 days/wk, 26 m/min, 20% grade). Evidence of training effect included a 5.9% increase in heart mass, increases in heart weight-to-body weight ratio, and a 60% increase in skeletal muscle citrate synthase activity in T rats compared with C rats. After the training program, cardiac myocytes were isolated from T and C hearts. Myocytes were chemically skinned (i.e., the sarcolemma was removed) and attached to a force transducer, and steady-state tension was determined in solutions of various Ca(2+) concentrations ([Ca(2+)]). Myocytes isolated from the hearts of T rats showed a significantly (P < 0.01) increased sensitivity of tension to [Ca(2+)]. The [Ca(2+)] giving 50% of maximal tension (pCa(50)) was 5.90 +/- 0.033 and 5.82 +/- 0.023 (SD) in T and C myocytes, respectively (n = 70 myocytes/group). This result suggests that exercise training affects the myofibrillar proteins, such that Ca(2+) sensitivity is increased, and that this may be the mechanism that underlies, at least in part, the effect of training to increase myocardial contractility.     

Influence of age and run training on cardiac Na+/Ca2+ exchange.

Effects of age and training on myocardial Na+/Ca2+ exchange were examined in young sedentary (YS; 14-15 mo), aged sedentary (AS; 27-31 mo), and aged trained (AT; 8- to 11-wk treadmill run training) male Fischer Brown Norway rats. Whole heart performance and isolated cardiocyte Na+/Ca2+ exchange characteristics were measured. At the whole heart level, a small but significant slowing of late isovolumic left ventricular (LV) relaxation, which may be indicative of altered Na+/Ca2+ exchange activity, was seen in hearts from AS rats. This subtle impairment in relaxation was not observed in hearts from AT rats. At the single-cardiocyte level, late action potential duration was prolonged, resting membrane potential was more positive, and overshoot potential was greater in cardiocytes from AS rats than from YS rats (P < 0.05). Training did not influence any of these age-related action potential characteristics. In electrically paced cardiocytes, neither shortening nor intracellular Ca2+ concentration ([Ca2+]i) dynamics was influenced by age or training. Similarly, neither age nor training influenced the rate of [Ca2+]i clearance via forward (Nain+ /Caout2+) Na+/Ca2+ exchange after caffeine-induced Ca2+ release from the sarcoplasmic reticulum or cardiac Na+/Ca2+ exchanger protein (NCX1) expression. However, when whole cell patch-clamp techniques combined with fluorescence microscopy were used to evaluate the ability of Na+/Ca2+ exchange to alter cytosolic [Ca2+] ([Ca2+]c) under conditions where membrane potential (Vm) and internal and external [Na+] and [Ca2+] could be controlled, we observed age-associated increases in forward Na+/Ca2+ exchange-mediated [Ca2+]c clearance (P < 0.05) that were not influenced by training. The age-related increase in forward Na+/Ca2+ exchange activity provides a hypothetical explanation for the late action potential prolongation observed in this study.     

Cardiac excitation-contraction coupling is altered in myocytes from aged male mice but not in cells from aged female mice

This study characterized age-related alterations in excitation-contraction (EC)-coupling in ventricular myocytes and investigated whether these alterations are affected by the sex of the animal. Voltage-clamp experiments were conducted in myocytes from young adult (approximately 7 mo) and aged (approximately 24 mo) male and female mice. Intracellular Ca(2+) concentrations and unloaded cell shortening were measured at 37 degrees C with fura-2 and a video edge detector. Fractional shortening and Ca(2+) current density were significantly reduced in aged male myocytes compared with those in young adult male cells. In addition, Ca(2+) transients were significantly smaller in aged male myocytes. Sarcoplasmic reticulum (SR) content, assessed by rapid application of 10 mM caffeine, declined with age in male myocytes. However, EC coupling gain and fractional release of SR Ca(2+) were similar in young adult and aged male cells. In contrast to results in male animals, fractional shortening and Ca(2+) current densities were similar in young adult and aged myocytes isolated from female hearts. Furthermore, Ca(2+) transient amplitudes were unaffected by age in female cells. Interestingly, SR Ca(2+) content was elevated in aged female myocytes, and fractional SR Ca(2+) release declined with age in females. However, the gain of EC coupling was not different in myocytes from young adult and aged female mice. These data demonstrate that age-related alterations in EC coupling are more prominent in myocytes from male hearts than in cells from female hearts and suggest that it is important to consider sex as a variable in studies of the effects of aging on cardiac EC coupling.     

Temperature effects on morphological integrity and Ca²⁺ signaling in freshly isolated murine feed artery endothelial cell tubes

To study Ca(2+) signaling in the endothelium of murine feed arteries, we determined the in vitro stability of endothelial cell (EC) tubes freshly isolated from abdominal muscle feed arteries of male and female C57BL/6 mice (5-9 mo, 25-35 g). We tested the hypothesis that intracellular Ca(2+) concentration ([Ca(2+)](i)) responses to muscarinic receptor activation would increase with temperature. Intact EC tubes (length: 1-2 mm, width: 65-80 μm) were isolated using gentle enzymatic digestion with trituration to remove smooth muscle cells. A freshly isolated EC tube was secured in a chamber and superfused at 24 (room temperature), 32, or 37°C. Using fura-2 dye, [Ca(2+)](i) was monitored (ratio of fluorescence at 340- to 380-nm wavelength) at rest and in response to bolus doses of ACh (20 nmol to 200 μmol). The morphological integrity of EC tubes was preserved at 24 and 32°C. Based on the Ca(2+) K(d) values we determined for fura-2 (174 nM at 24°C and 146 nM at 32°C), resting [Ca(2+)](i) remained stable for 180 min at both 24 and 32°C (27 ± 4 and 34 ± 2 nM, respectively), with peak responses to ACh (20 μmol) increasing from ～220 nM at 24°C to ～500 nM at 32°C (P < 0.05). There was no difference in responses to ACh between EC tubes from male versus female mice. When EC tubes were maintained at 37°C (typical in vivo temperature), resting [Ca(2+)](i) increased by ～30% within 15 min, and gaps formed between individual ECs as they retracted and extruded dye, precluding further study. We conclude that EC tubes enable Ca(2+) signaling to be evaluated in the freshly isolated endothelium of murine feed arteries. While Ca(2+) responses are enhanced by approximately twofold at 32 versus 24°C, the instability of EC tubes at 37°C precludes their study at typical body temperature.     

Low‐intensity aerobic exercise improves cardiac remodelling of adult spontaneously hypertensive rats

We evaluated the influence of aerobic training on cardiac remodeling in untreated spontaneously hypertensive rats (SHR). Four experimental groups were used: sedentary (W‐SED, n=27) and trained (WEX, n=31) normotensive Wistar rats, and sedentary (SHR‐SED, n=27) and exercised (SHR‐EX, n=32) hypertensive rats. At 13 months old, trained groups underwent treadmill exercise five days a week for four months. Statistical analysis: ANOVA or Kruskal‐Wallis. Exercised groups had higher physical capacity. Hypertensive groups presented left ventricular (LV) concentric hypertrophy with impaired function. Left atrium diameter, LV posterior wall thickness and relative thickness, and isovolumetric relaxation time were lower in SHR‐EX than SHR‐SED. Interstitial collagen fraction and Type I‐Type III collagen ratio were higher in SHR‐SED than W‐SED. In SHR‐EX these parameters had intermediate values between W‐EX and SHRSED with no differences between either group. Myocardial matrix metalloproteinase‐2 activity, evaluated by zymography, was higher in SHR‐SED than W‐SED and SHR‐EX. TIMP‐2 was higher in hypertensive than normotensive groups. In conclusion, low intensity aerobic exercise reduces left atrium dimension and LV posterior wall thickness, and improves functional capacity, diastolic function, and metalloproteinase‐2 activity in adult SHR.     

Separate and combined effects of exercise training and weight loss on exercise efficiency and substrate oxidation

Perturbations in body weight have been shown to affect energy expenditure and efficiency during physical activity. The separate effects of weight loss and exercise training on exercise efficiency or the proportion of energy derived from fat oxidation during physical activity, however, are not known. The purpose of this study was to determine the separate and combined effects of exercise training and weight loss on metabolic efficiency, economy (EC), and fat oxidation during steady-state moderate submaximal exercise. Sixty-four sedentary older (67 +/- 0.5 yr) overweight to obese (30.7 +/- 0.4 kg/m(2)) volunteers completed 4 mo of either diet-induced weight loss (WL; n = 11), exercise training (EX; n = 36), or the combination of both interventions (WLEX; n = 17). Energy expenditure, gross efficiency (GE), EC, and proportion of energy expended from fat (EF) were determined during a 1-h submaximal (50% of peak aerobic capacity) cycle ergometry exercise before the intervention and at the same absolute work rate after the intervention. We found that EX increased GE by 4.7 +/- 2.2%. EC was similarly increased by 4.2 +/- 2.1% by EX. The addition of concomitant WL to EX (WLEX) resulted in greater increases in GE (9.0 +/- 3.3%) compared with WL alone but not compared with EX alone. These effects remained after adjusting for changes in lean body mass. The proportion of energy derived from fat during the bout of moderate exercise increased with EX and WLEX but not with WL. From these findings, we conclude that exercise training, either alone or in combination with weight loss, increases both exercise efficiency and the utilization of fat during moderate physical activity in previously sedentary, obese older adults. Weight loss alone, however, significantly improves neither efficiency nor utilization of fat during exercise.     

Ca²⁺ sensitization of cardiac myofilament proteins contributes to exercise training-enhanced myocardial function in a porcine model of chronic occlusion

Exercise training has been shown to improve cardiac dysfunction in both patients and animal models of coronary artery disease; however, the underlying cellular and molecular mechanisms have not been completely understood. We hypothesized that exercise training would improve force generation in the myocardium distal to chronic coronary artery occlusion via altered intracellular Ca(2+) concentration ([Ca(2+)](i)) cycling and/or Ca(2+) sensitization of myofilaments. Ameroid occluders were surgically placed around the proximal left circumflex coronary artery of adult female Yucatan pigs. Twenty-two weeks postoperatively, the myocardium was isolated from nonoccluded (left anterior descending artery dependent) and collateral-dependent (formerly left circumflex coronary artery dependent) regions of sedentary (pen confined) and exercise-trained (treadmill run, 5 days/wk for 14 wk) pigs. Force measurements in myocardial strips showed that the percent change in force at stimulation frequencies of 3 and 4 Hz relative to 1 Hz was significantly higher in exercise-trained pigs compared with sedentary pigs. β-Adrenergic stimulation with dobutamine significantly improved force kinetics in myocardial strips of sedentary but not exercise-trained pigs at 1 Hz. Additionally, time to peak and half-decay of intracellular Ca(2+) (340-to-380-nm fluoresence ratio) responses at 1 Hz were significantly decreased in the collateral-dependent region of exercise-trained pigs with no difference in peak [Ca(2+)](i) between groups. Furthermore, the skinned myocardium from exercise-trained pigs showed an increase in Ca(2+) sensitivity compared with sedentary pigs. Immunoblot analysis revealed that the relative levels of cardiac troponin T and β(1)-adrenergic receptors were decreased in hearts from exercise-trained pigs independent of occlusion. Also, the ratio of phosphorylated to total myosin light chain-2, basal phosphorylation levels of cardiac troponin I (Ser(23) and Ser(24)), and cardiac myosin binding protein-C (Ser(282)) were unaltered by occlusion or exercise training. Thus, our data demonstrate that exercise training-enhanced force generation in the nonoccluded and collateral-dependent myocardium was associated with improved Ca(2+) transients, increased Ca(2+) sensitization of myofilament proteins, and decreased expression levels of β(1)-adrenergic receptors and cardiac troponin T.     

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.     

Exercise training alters length dependence of contractile properties in rat myocardium.

Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. Exercise training increases the sensitivity of rat cardiac myocytes to activation by Ca(2+), and this Ca(2+) sensitivity has been shown to be highly dependent on sarcomere length. We tested the hypothesis that exercise training increases this length dependence in cardiac myocytes. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise-trained (T) groups. The T rats underwent 11 wk of progressive treadmill exercise. Heart weight increased by 14% in T compared with C rats, and plantaris muscle citrate synthase activity showed a 39% increase with training. Steady-state tension was determined in permeabilized myocytes by using solutions of various Ca(2+) concentration (pCa), and tension-pCa curves were generated at two different sarcomere lengths for each myocyte (1.9 and 2.3 microm). We found an increased sarcomere length dependence of both maximal tension and pCa(50) (the Ca(2+) concentration giving 50% of maximal tension) in T compared with C myocytes. The DeltapCa(50) between the long and short sarcomere length was 0.084 +/- 0.023 (mean +/- SD) in myocytes from C hearts compared with 0.132 +/- 0.014 in myocytes from T hearts (n = 50 myocytes per group). The Deltamaximal tension was 5.11 +/- 1.42 kN/m(2) in C myocytes and 9.01 +/- 1.28 in T myocytes. We conclude that exercise training increases the length dependence of maximal and submaximal tension in cardiac myocytes, and this change may underlie, at least in part, training-induced enhancement of myocardial function.     

Alterations in PKC signaling underlie enhanced myogenic tone in exercise-trained porcine coronary resistance arteries.

The intracellular mechanisms underlying enhanced myogenic contraction (MC) in coronary resistance arteries (CRAs) from exercise-trained (EX) pigs have not been established. The purpose of this study was to test the hypothesis that exercise-induced alterations in protein kinase C (PKC) signaling underlie enhanced MC. Furthermore, we sought to determine whether modulation of intracellular Ca(2+) signaling by PKC underlies enhanced MC in EX animals. Male Yucatan miniature swine were treadmill trained (n = 7) at approximately 75% of maximal O(2) uptake for 16 wk (6 miles/h, 60 min) or remained sedentary (SED, n = 6). Diameter measurements in response to intraluminal pressure (60, 75, and 90 cmH(2)O) or 60 mM KCl were determined in single, cannulated CRAs ( approximately 100 microm ID) with and without the PKC inhibitor chelerythrine (CE, 1 microM). Confocal imaging of Ca(2+) signaling [myogenic Ca(2+) (Ca(m))] was also performed in CRAs of similar internal diameter after abluminal loading of the Ca(2+) indicator dye fluo 4 (1 microM, 37 degrees C, 30 min). We observed significantly greater MC in CRAs isolated from EX than from SED animals at 90 cmH(2)O, as well as greater reductions in MC after CE at all pressures studied. At intraluminal pressures of 75 and 90 cmH(2)O, CE produced greater decreases in Ca(m) in CRAs from EX than from SED animals (64% vs. 25%, P < 0.05). Inhibition of KCl constriction and Ca(m) by CE was also greater in EX animals (P < 0.05). Western blotting revealed significant increases in Ca(2+)-dependent PKC-alpha ( approximately 50%) but not Ca(2+)-independent PKC-epsilon levels in CRAs isolated from EX animals (P < 0.05). We also observed significant group differences in phosphorylated PKC-alpha levels. Finally, voltage-gated Ca(2+) current (VGCC) was effectively blocked by CE, bisindolylmaleimide, and staurosporine in isolated smooth muscle cells from CRAs, providing evidence for a mechanistic link between VGCCs and PKC in our experimental paradigm. These results suggest that enhanced MC in CRAs from EX animals involves PKC-dependent modulation of intracellular Ca(2+), including regulation of VGCCs.     

The II-III loop of the skeletal muscle dihydropyridine receptor is responsible for the Bi-directional coupling with the ryanodine receptor.

The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca(2+) release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca(2+) current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca(2+) currents. Introducing a short skeletal segment (alpha(1S) residues 720-765) into the cardiac II-III loop (replacing alpha(1C) residues 851-896) of GFP-SkLC restored both EC coupling and Ca(2+) current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem. 273, 24983-24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca(2+) current.     

Differential interactions of Na+ channel toxins with T-type Ca2+ channels

Two types of voltage-dependent Ca(2+) channels have been identified in heart: high (I(CaL)) and low (I(CaT)) voltage-activated Ca(2+) channels. In guinea pig ventricular myocytes, low voltage-activated inward current consists of I(CaT) and a tetrodotoxin (TTX)-sensitive I(Ca) component (I(Ca(TTX))). In this study, we reexamined the nature of low-threshold I(Ca) in dog atrium, as well as whether it is affected by Na(+) channel toxins. Ca(2+) currents were recorded using the whole-cell patch clamp technique. In the absence of external Na(+), a transient inward current activated near -50 mV, peaked at -30 mV, and reversed around +40 mV (HP = -90 mV). It was unaffected by 30 microM TTX or micromolar concentrations of external Na(+), but was inhibited by 50 microM Ni(2+) (by approximately 90%) or 5 microM mibefradil (by approximately 50%), consistent with the reported properties of I(CaT). Addition of 30 microM TTX in the presence of Ni(2+) increased the current approximately fourfold (41% of control), and shifted the dose-response curve of Ni(2+) block to the right (IC(50) from 7.6 to 30 microM). Saxitoxin (STX) at 1 microM abolished the current left in 50 microM Ni(2+). In the absence of Ni(2+), STX potently blocked I(CaT) (EC(50) = 185 nM) and modestly reduced I(CaL) (EC(50) = 1.6 microM). While TTX produced no direct effect on I(CaT) elicited by expression of hCa(V)3.1 and hCa(V)3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni(2+) (IC(50) increased to 550 microM Ni(2+) for Ca(V)3.1 and 15 microM Ni(2+) for Ca(V)3.2); in contrast, 30 microM TTX directly inhibited hCa(V)3.3-induced I(CaT) and the addition of 750 microM Ni(2+) to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni(2+) alone. 1 microM STX directly inhibited Ca(V)3.1-, Ca(V)3.2-, and Ca(V)3.3-mediated I(CaT) but did not enhance the ability of Ni(2+) to block these currents. These findings provide important new implications for our understanding of structure-function relationships of I(CaT) in heart, and further extend the hypothesis of a parallel evolution of Na(+) and Ca(2+) channels from an ancestor with common structural motifs.     

Swimming training promotes cardiac remodeling and alters the expression of mRNA and protein levels involved in calcium handling in hypertensive rats

Aim

The aim of this study was to identify the effects of swimming training on the mRNA expression and protein levels of the calcium handling proteins in the hearts of renovascular hypertensive rats submitted to swimming protocol during 6 weeks.

Main methods

Fischer rats with renovascular hypertension 2-kidney 1-clip (2K1C) and SHAM groups were divided among sedentary and exercised groups. The exercise protocol lasted for 6 weeks (1 h/day, 5×/week), and the mean arterial pressure, cardiomyocytes hypertrophy parameters, mRNA expression and protein levels of some calcium handling proteins in the left ventricle were evaluated.

Key findings

Swimming training was able to reduce the levels of mean arterial pressure in the hypertensive group compared to 2K1C SED, and to promote cardiac hypertrophy in SHAM EX and 2K1C EX groups in comparison to the respective control groups. The mRNA levels of B-type natriuretic peptide were reduced in the 2K1C EX when compared to 2K1C SED. The mRNA and protein levels of the sarcoplasmic reticulum Ca^2 +-ATPase increased after the swimming training in SHAM and 2K1C groups. The mRNA and protein levels of phospholamban, displayed an increase in their levels in the exercised SHAM and in hypertensive rats in comparison to their respective controls; while mRNA levels of Na⁺/Ca^2 + exchanger was reduced in the left ventricle comparing to the sedentary hypertensive rats.

Significance

Taken altogether, we provide evidence that the aerobic training may lead to cardiac remodeling, and modulate the calcium handling proteins expression in the heart of hypertensive rats.     

Mutation of the calmodulin binding motif IQ of the L-type Ca(v)1.2 Ca2+ channel to EQ induces dilated cardiomyopathy and death

Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca(2+)/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Ca(v)1.2 channel and induces Ca(2+)-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Ca(v)1.2(I1624E) mutation were generated in adult heterozygous mice through inactivation of the floxed WT Ca(v)1.2(L2) allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Ca(v)1.2(I1624E) hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Ca(v)1.2(I1624E) channel protein and I(Ca). The Ca(v)1.2(I1624E) channel showed "CDI" kinetics. Despite a lower sarcoplasmic reticulum Ca(2+) content, cellular contractility and global Ca(2+) transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Ca(v)1.2(I1624E) hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.