S100A1: A regulator of myocardial contractility

S100A1, a Ca(2+) binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been found to colocalize with the sarcoplasmic reticulum (SR) and the contractile filaments in cardiac tissue. Because S100A1 is known to modulate SR Ca(2+) handling in skeletal muscle, we sought to investigate the specific role of S100A1 in the regulation of myocardial contractility. To address this issue, we investigated contractile properties of adult cardiomyocytes as well as of engineered heart tissue after S100A1 adenoviral gene transfer. S100A1 gene transfer resulted in a significant increase of unloaded shortening and isometric contraction in isolated cardiomyocytes and engineered heart tissues, respectively. Analysis of intracellular Ca(2+) cycling in S100A1-overexpressing cardiomyocytes revealed a significant increase in cytosolic Ca(2+) transients, whereas in functional studies on saponin-permeabilized adult cardiomyocytes, the addition of S100A1 protein significantly enhanced SR Ca(2+) uptake. Moreover, in Triton-skinned ventricular trabeculae, S100A1 protein significantly decreased myofibrillar Ca(2+) sensitivity ([EC(50%)]) and Ca(2+) cooperativity, whereas maximal isometric force remained unchanged. Our data suggest that S100A1 effects are cAMP independent because cellular cAMP levels and protein kinase A-dependent phosphorylation of phospholamban were not altered, and carbachol failed to suppress S100A1 actions. These results show that S100A1 overexpression enhances cardiac contractile performance and establish the concept of S100A1 as a regulator of myocardial contractility. S100A1 thus improves cardiac contractile performance both by regulating SR Ca(2+) handling and myofibrillar Ca(2+) responsiveness.     

The Na+/Ca2+ exchange blocker SEA0400 fails to enhance cytosolic Ca2+ transient and contractility in canine ventricular cardiomyocytes

AIMS: This study was designed to evaluate the effects of the Na(+)/Ca(2+) exchange (NCX) inhibitor SEA0400 on Ca(2+) handling in isolated canine ventricular myocytes. METHODS AND RESULTS: Intracellular Ca(2+) ([Ca(2+)](i)) transients, induced by either field stimulation or caffeine flush, were monitored using Ca(2+) indicator dyes. [Ca(2+)](i)-dependent modulation of the inhibitory effect of SEA0400 on NCX was characterized by the changes in Ni(2+)-sensitive current in voltage-clamped myocytes. Sarcoplasmic reticulum (SR) Ca(2+) release and uptake were studied in SR membrane vesicles. Gating properties of single-ryanodine receptors were analysed in lipid bilayers. Ca(2+) sensitivity of the contractile machinery was evaluated in chemically skinned myocytes. In myocytes paced at 1 Hz, neither diastolic [Ca(2+)](i) nor the amplitude of [Ca(2+)](i) transients was significantly altered by SEA0400 up to the concentration of 1 microM, which was shown to inhibit the exchange current. The blocking effect of SEA0400 on NCX decreased with increasing [Ca(2+)](i), and it was more pronounced in reverse than in forward mode operation at every [Ca(2+)](i) examined. The rate of decay of the caffeine-induced [Ca(2+)](i) transients was decreased significantly by 1 microM SEA0400; however, this effect was only a fraction of that observed with 10 mM NiCl(2). Neither SR Ca(2+) release and uptake nor cell shortening and Ca(2+) sensitivity of the contractile proteins were influenced by SEA0400. CONCLUSION: The lack of any major SEA0400-induced shift in Ca(2+) transients or contractility of myocytes can well be explained by its limited inhibitory effect on NCX (further attenuated by elevated [Ca(2+)](i) levels) and a concomitant reduction in Ca(2+) influx due to the predominantly reverse mode blockade of NCX and suppression of L-type Ca(2+) current.     

PI3Kgamma is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes

We recently showed that phosphoinositide-3-kinase-gamma-deficient (PI3Kgamma(-/-)) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca(2+) content and release but not L-type Ca(2+) current (I(Ca,L)), demonstrating PI3Kgamma locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3Kgamma ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca(2+) transients, SR Ca(2+) content, and phospholamban phosphorylation (PLN-PO(4)) by similar amounts to levels observed in untreated PI3Kgamma(-/-) myocytes. Combined PDE3 and PDE4 inhibition caused no further increases in SR function. By contrast, only PDE3 inhibition affected Ca(2+) transients, SR Ca(2+) loads, and PLN-PO(4) levels in PI3Kgamma(-/-) myocytes. On the other hand, inhibition of PDE3 or PDE4 alone did not affect I(Ca,L) in either PI3Kgamma(-/-) or WT cardiomyocytes, whereas simultaneous PDE3 and PDE4 inhibition elevated I(Ca,L) in both groups. Ryanodine receptor (RyR(2)) phosphorylation levels were not different in basal conditions between PI3Kgamma(-/-) and WT myocytes and increased in both groups with PDE inhibition. Our results establish that L-type Ca(2+) channels, RyR(2), and SR Ca(2+) pumps are regulated differently in distinct subcellular compartments by PDE3 and PDE4. In addition, the loss of PI3Kgamma selectively abolishes PDE4 activity, not PDE3, in subcellular compartments containing the SR Ca(2+)-ATPase but not RyR(2) or L-type Ca(2+) channels.     

Cardiac unloading alters contractility and calcium homeostasis in ventricular myocytes

Altered cardiac workload has an important effect on myocyte structure and function. Cardiac hypertrophy resulting from an increase in load has been studied extensively in the past. However, the effects of unloading and atrophy have recently become of more interest since devices for mechanical left ventricular unloading have been introduced into clinical practice for the treatment of patients with terminal heart failure, and a resulting improved cardiac and myocyte contractility have been reported. We used the heterotopic abdominal mouse heart transplant model in order to study the effects of 5 days of unloading on cell size (confocal microscopy), contractility (fractional shortening: video motion), calcium homeostasis ([Ca(2+)](i)transients, SR Ca(2+)content); and L-type Ca(2+)and sodium/calcium exchanger currents (whole cell patch clamp technique). We found unloading caused decreased cell volume consistent with atrophy. An increased fractional shortening and [Ca(2+)](i)transient were observed in myocytes from unloaded hearts as compared with controls. Transsarcolemmal I(Ca,L)and I(Na/Ca)densities, and SR Ca(2+)content were unaltered, as was membrane capacitance. A reduction in cell volume with mainteinance of internal and surface membrane areas, and/or a decrease in concentration of cellular protein Ca(2+)buffers, may contribute to the increase in the [Ca(2+)](i)transient in this model.     

Cellular basis of abnormal calcium transients of failing human ventricular myocytes

Depressed contractility is a central feature of the failing human heart and has been attributed to altered [Ca2+]i. This study examined the respective roles of the L-type Ca2+ current (ICa), SR Ca2+ uptake, storage and release, Ca2+ transport via the Na+-Ca2+ exchanger (NCX), and Ca2+ buffering in the altered Ca2+ transients of failing human ventricular myocytes. Electrophysiological techniques were used to measure and control V(m) and measure I(m), respectively, and Fluo-3 was used to measure [Ca2+]i in myocytes from nonfailing (NF) and failing (F) human hearts. Ca2+ transients from F myocytes were significantly smaller and decayed more slowly than those from NF hearts. Ca2+ uptake rates by the SR and the amount of Ca2+ stored in the SR were significantly reduced in F myocytes. There were no significant changes in the rate of Ca2+ removal from F myocytes by the NCX, in the density of NCX current as a function of [Ca2+]i, ICa density, or cellular Ca2+ buffering. However, Ca2+ influx during the late portions of the action potential seems able to elevate [Ca2+]i in F but not in NF myocytes. A reduction in the rate of net Ca2+ uptake by the SR slows the decay of the Ca2+ transient and reduces SR Ca2+ stores. This leads to reduced SR Ca2+ release, which induces additional Ca2+ influx during the plateau phase of the action potential, further slowing the decay of the Ca2+ transient. These changes can explain the defective Ca2+ transients of the failing human ventricular myocyte.     

Contractile reserve and intracellular calcium regulation in mouse myocytes from normal and hypertrophied failing hearts

Mouse myocyte contractility and the changes induced by pressure overload are not fully understood. We studied contractile reserve in isolated left ventricular myocytes from mice with ascending aortic stenosis (AS) during compensatory hypertrophy (4-week AS) and the later stage of early failure (7-week AS) and from control mice. Myocyte contraction and [Ca(2+)](i) transients with fluo-3 were measured simultaneously. At baseline (0.5 Hz, 1.5 mmol/L [Ca(2+)](o), 25 degrees C), the amplitude of myocyte shortening and peak-systolic [Ca(2+)](i) in 7-week AS were not different from those of controls, whereas contraction, relaxation, and the decline of [Ca(2+)](i) transients were slower. In response to the challenge of high [Ca(2+)](o), fractional cell shortening was severely depressed with reduced peak-systolic [Ca(2+)](i) in 7-week AS compared with controls. In response to rapid pacing stimulation, cell shortening and peak-systolic [Ca(2+)](i) increased in controls, but this response was depressed in 7-week AS. In contrast, the responses to both challenge with high [Ca(2+)](o) and rapid pacing in 4-week AS were similar to those of controls. Although protein levels of Na(+)-Ca(2+) exchanger were increased in both 4-week and 7-week AS, the ratio of SR Ca(2+)-ATPase to phospholamban protein levels was depressed in 7-week AS compared with controls but not in 4-week AS. This was associated with an impaired capacity to increase sarcoplasmic reticulum Ca(2+) load during high work states in 7-week AS myocytes. In hypertrophied failing mouse myocytes, depressed contractile reserve is related to an impaired augmentation of systolic [Ca(2+)](i) and SR Ca(2+) load and simulates findings in human failing myocytes.     

Altered myocardial Ca2+ cycling after left ventricular assist device support in the failing human heart

OBJECTIVES: The objective of the present study was to determine whether improved contractility after left ventricular assist device (LVAD) support reflects altered myocyte calcium cycling and changes in calcium-handling proteins. BACKGROUND: Previous reports demonstrate that LVAD support induces sustained unloading of the heart with regression of pathologic hypertrophy and improvements in contractile performance. METHODS: In the human myocardium of subjects with heart failure (HF), with non-failing hearts (NF), and with LVAD-supported failing hearts (HF-LVAD), intracellular calcium ([Ca(2+)](i)) transients were measured in isolated myocytes at 0.5 Hz, and frequency-dependent force generation was measured in multicellular preparations (trabeculae). Abundance of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA), Na(+)/Ca(2+) exchanger (NCX), and phospholamban was assessed by Western analysis. RESULTS: Compared with NF myocytes, HF myocytes exhibited a slowed terminal decay of the Ca(2+) transient (DT(terminal), 376 +/- 18 ms vs. 270 +/- 21 ms, HF vs. NF, p < 0.0008), and HF-LVAD myocytes exhibited a DT(terminal) that was much shorter than that observed in HF myocytes (278 +/- 10 ms, HF vs. HF-LVAD, p < 0.0001). Trabeculae from HF showed a negative force-frequency relationship, compared with a positive relationship in NF, whereas a neutral relationship was observed in HF-LVAD. Although decreased SERCA abundance in HF was not altered by LVAD support, improvements in [Ca(2+)](i) transients and frequency-dependent contractile function were associated with a significant decrease in NCX abundance and activity from HF to HF-LVAD. CONCLUSIONS: Improvement in rate-dependent contractility in LVAD-supported failing human hearts is associated with a faster decay of the myocyte calcium transient. These improvements reflect decreases in NCX abundance and transport capacity without significant changes in SERCA after LVAD support. Our results suggest that reverse remodeling may involve selective, rather than global, normalization of the pathologic patterns associated with the failing heart.     

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.     

Cardiac sarcoplasmic reticulum calcium release and load are enhanced by subcellular cAMP elevations in PI3Kgamma-deficient mice

We recently showed that phosphoinositide-3-kinase-gamma-deficient (PI3Kgamma-/-) mice have increased cardiac contractility without changes in heart size compared with control mice (ie, PI3Kgamma+/+ or PI3Kgamma+/-). In this study, we show that PI3Kgamma-/- cardiomyocytes have elevated Ca2+ transient amplitudes with abbreviated decay kinetics compared with control under field-stimulation and voltage-clamp conditions. When Ca2+ transients were eliminated with high Ca2+ buffering, L-type Ca2+ currents (I(Ca,L)), K+ currents, and action potential duration (APD) were not different between the groups, whereas, in the presence of Ca2+ transients, Ca2+-dependent phase of I(Ca,L) inactivation was abbreviated and APD at 90% repolarization was prolonged in PI3Kgamma-/- mice. Excitation-contraction coupling (ECC) gain, sarcoplasmic reticulum (SR) Ca2+ load, and SR Ca(2+) release fluxes measured as Ca2+ spikes, were also increased in PI3Kgamma-/- cardiomyocytes without detectable changes in Ca2+ spikes kinetics. The cAMP inhibitor Rp-cAMP eliminated enhanced ECC and SR Ca2+ load in PI3Kgamma-/- without effects in control myocytes. On the other hand, the beta-adrenergic receptor agonist isoproterenol increased I(Ca,L) and Ca2+ transient equally by approximately 2-fold in both PI3Kgamma-/- and PI3Kgamma+/- cardiomyocytes. Our results establish that PI3Kgamma reduces cardiac contractility in a highly compartmentalized manner by inhibiting cAMP-mediated SR Ca2+ loading without directly affecting other major modulators of ECC, such as AP and I(Ca,L).     

Preserved cardiomyocyte function and altered desmin pattern in transgenic mouse model of dilated cardiomyopathy

Taking advantage of the unique model of slowly developing dilated cardiomyopathy in mice with cardiomyocyte-specific transgenic overexpression of activated Gαq protein (Tgαq*44 mice) we analyzed the contribution of the cardiomyocyte malfunction, fibrosis and cytoskeleton remodeling to the development of heart failure in this model. Left ventricular (LV) in vivo function, myocardial fibrosis, cytoskeletal proteins expression and distribution, Ca(2+) handling and contractile function of isolated cardiomyocytes were evaluated at the stages of the early, compensated, and late, decompensated heart failure in 4-, 12- and 14-month-old Tgαq*44 mice, respectively, and compared to age-matched wild-type FVB mice. In the 4-month-old Tgαq*44 mice significant myocardial fibrosis, moderate myocyte hypertrophy and increased expression of regularly arranged and homogenously distributed desmin accompanied by increased phosphorylation of desmin chaperone protein, αB-crystallin, were found. Cardiomyocyte shortening, Ca(2+) handling and LV function were not altered. At 12 and 14 months of age, Tgαq*44 mice displayed progressive deterioration of the LV function. The contractile performance of isolated myocytes was still preserved, and the amplitude of Ca(2+) transients was even increased probably due to impairment of Na(+)/Ca(2+) exchanger function, while fibrosis was more extensive than in younger mice. Moreover, substantial disarrangement of desmin distribution accompanied by decreasing phosphorylation of αB-crystallin appeared. In Tgαq*44 mice disarrangement of desmin, at least partly related to inadequate phosphorylation of αB-crystallin seems to be importantly involved in the progressive deterioration of contractile heart function.     

Adenoviral gene transfer of mutant phospholamban rescues contractile dysfunction in failing rabbit myocytes with relatively preserved SERCA function

In heart failure (HF) a main factor in reduced contractility is reduced SR Ca2+ content and reversed force-frequency response (FFR), ie, from positive to negative. Our arrhythmogenic rabbit HF model exhibits decreased contractility mainly due to an increase in Na/Ca exchange (NCX) activity (with only modest decrease in SR Ca2+-ATPase (SERCA) function), similar to many end-stage HF patients. Here we test whether phospholamban (PLB) inhibition using a dominant-negative mutant PLB adenovirus (K3E/R14E, AdPLB-dn, with beta-galactosidase adenovirus as control) could enhance SERCA function and restore Ca2+ transients and positive FFR in ventricular myocytes from these HF rabbits. HF myocytes infected with AdPLB-dn (versus control) had enhanced Ca2+ transient amplitude (2.0+/-0.1 versus 1.6+/-0.05 F/Fo at 0.5 Hz, P<0.05) and had a positive FFR, whereas acutely isolated HF myocytes or those infected with Adbetagal had negative FFR. Ca2+ transients declined faster in AdPLB-dn versus Adbetagal myocytes (RT50%: 317+/-29 versus 551+/-90 ms at 0.5 Hz, P<0.05) and had an increased SR Ca2+ load (3.5+/-0.3 versus 2.6+/-0.2 F/Fo at 0.5 Hz, P<0.05), indicative of increased SERCA function. Furthermore, this restoration of function was not due to changes in NCX or SERCA expression. Thus, increasing SERCA activity in failing myocytes by AdPLB-dn gene transfer reversed the contractile dysfunction (and restored positive FFR) by increasing SR Ca2+ load. This approach could enhance contractile function in failing hearts of various etiologies, even here where reduced SERCA activity is not the main dysfunction.     

The Na+/Ca2+ exchanger/SR Ca2+ ATPase transport capacity regulates the contractility of normal and hypertrophied feline ventricular myocytes

BACKGROUND: Pressure overload leads to cardiac hypertrophy, which is often followed by heart failure. We tested the hypothesis that depressed contractility in this process results from an imbalance in Ca 2+ transport by the sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) and the sarcolemmal Na+/Ca2+ exchanger (NCX). METHODS AND RESULTS: Left ventricular (LV) myocytes (n = 79) from 12 normal (N) and 5 hypertrophied (LVH, by aortic banding) feline hearts were studied. Adenoviral gene transfer was used to introduce green fluorescent protein (GFP), SERCA2, and NCX into N and LVH myocytes. Contraction (videomicroscopy) and Ca2+ transients (Fluo-3) were measured in steady state and after rest periods of 2 to 120 seconds (rest decay and potentiation). LVH hearts were significantly larger than N (7.1 +/- 1.4 versus 4.2 +/- 0.2 g/kg). SERCA protein was significantly less abundant in LVH versus N. Steady state contractions and Ca2+ transients of LVH-GFP myocytes decayed more slowly and rest decay of contractility was more pronounced compared with N-GFP. Infection of LVH (and N) myocytes with SERCA increased basal contractility and reduced rest decay. Infection of LVH myocytes with NCX almost abolished contraction and in N myocytes reduced contractility and increased rest decay. CONCLUSION: These findings suggest that an imbalance of Ca2+ transport by SERCA and the NCX produces the characteristic contractile abnormalities of hypertrophied cardiac myocytes.     

Abnormalities of calcium cycling in the hypertrophied and failing heart

Progressive deterioration of cardiac contractility is a central feature of congestive heart failure (CHF) in humans. In this report we review those studies that have addressed the idea that alterations of intracellular calcium (Ca(2+)) regulation is primarily responsible for the depressed contractility of the failing heart. The review points out that Ca(2+)transients and contraction are similar in non-failing and failing myocytes at very slow frequencies of stimulation (and other low stress environments). Faster pacing rates, high Ca(2+)and beta-adrenergic stimulation reveal large reductions in contractile reserve in failing myocytes. The underlying cellular basis of these defects is then considered. Studies showing changes in the abundance of L-type Ca(2+)channels, Ca(2+)transport proteins [sarcoplasmic reticulum Ca(2+)ATPase (SERCA2), phospholamban (PLB), Na(+)/Ca(2+) exchanger (NCX)] and Ca(2+) release channels (RYR) in excitation-contraction coupling and Ca(2+)release and uptake by the sarcoplasmic reticulum (SR) are reviewed. These observations support our hypotheses that (i) defective Ca(2+)regulation involves multiple molecules and processes, not one molecule, (ii) the initiation and progression of CHF inolves defective Ca(2+)regulation, and (iii) prevention or correction of Ca(2+)regulatory defects in the early stages of cardiac diseases can delay or prevent the onset of CHF.     

Familial hypertrophic cardiomyopathy-linked mutant troponin T causes stress-induced ventricular tachycardia and Ca2+-dependent action potential remodeling

The cardiac troponin T (TnT) I79N mutation has been linked to familial hypertrophic cardiomyopathy and high incidence of sudden death, despite causing little or no cardiac hypertrophy in patients. Transgenic mice expressing mutant human TnT (I79N-Tg) have increased cardiac contractility, but no ventricular hypertrophy or fibrosis. Enhanced cardiac function has been associated with myofilament Ca2+ sensitization, suggesting altered cellular Ca2+ handling. In the present study, we compare cellular Ca2+ transients and electrophysiological parameters of 64 I79N-Tg and 106 control mice in isolated myocytes, isolated perfused hearts, and whole animals. Ventricular action potentials (APs) measured in isolated I79N-Tg hearts and myocytes were significantly shortened only at 70% repolarization. No significant differences were found either in L-type Ca2+ or transient outward K+ currents, but inward rectifier K+ current (IK1) was significantly decreased. More critically, Ca2+ transients of field-stimulated ventricular I79N-Tg myocytes were reduced and had slow decay kinetics, consistent with increased Ca2+ sensitivity of I79N mutant fibers. AP differences were abolished when myocytes were dialyzed with Ca2+ buffers or after the Na+-Ca2+ exchanger was blocked by Li+. At higher pacing rates or in presence of isoproterenol, diastolic Ca2+ became significantly elevated in I79N-Tg compared with control myocytes. Ventricular ectopy could be induced by isoproterenol-challenge in isolated I79N-Tg hearts and anesthetized I79N-Tg mice. Freely moving I79N-Tg mice had a higher incidence of nonsustained ventricular tachycardia (VT) during mental stress (warm air jets). We conclude that the TnT-I79N mutation causes stress-induced VT even in absence of hypertrophy and/or fibrosis, arising possibly from the combination of AP remodeling related to altered Ca2+ transients and suppression of IK1.     

Ca2+ influx through T- and L-type Ca2+ channels have different effects on myocyte contractility and induce unique cardiac phenotypes

T-type Ca(2+) channels (TTCCs) are expressed in the developing heart, are not present in the adult ventricle, and are reexpressed in cardiac diseases involving cardiac dysfunction and premature, arrhythmogenic death. The goal of this study was to determine the functional role of increased Ca(2+) influx through reexpressed TTCCs in the adult heart. A mouse line with cardiac-specific, conditional expression of the alpha1G-TTCC was used to increase Ca(2+) influx through TTCCs. alpha1G hearts had mild increases in contractility but no cardiac histopathology or premature death. This contrasts with the pathological phenotype of a previously studied mouse with increased Ca(2+) influx through the L-type Ca(2+) channel (LTCC) secondary to overexpression of its beta2a subunit. Although alpha1G and beta2a myocytes had similar increases in Ca(2+) influx, alpha1G myocytes had smaller increases in contraction magnitude, and, unlike beta2a myocytes, there were no increases in sarcoplasmic reticulum Ca(2+) loading. Ca(2+) influx through TTCCs also did not induce normal sarcoplasmic reticulum Ca(2+) release. alpha1G myocytes had changes in LTCC, SERCA2a, and phospholamban abundance, which appear to be adaptations that help maintain Ca(2+) homeostasis. Immunostaining suggested that the majority of alpha1G-TTCCs were on the surface membrane. Osmotic shock, which selectively eliminates T-tubules, induced a greater reduction in L- versus TTCC currents. These studies suggest that T- and LTCCs are in different portions of the sarcolemma (surface membrane versus T-tubules) and that Ca(2+) influx through these channels induce different effects on myocyte contractility and lead to distinct cardiac phenotypes.     

Calcitriol modulation of cardiac contractile performance via protein kinase C

Vitamin D(3) deficiency enhances cardiac contraction in experimental studies, yet paradoxically this deficiency is linked to congestive heart failure in humans. Activated vitamin D(3) (1alpha,25-dihydroxyvitamin D(3)) or calcitriol, decreases peak force and activates protein kinase C (PKC) in isolated perfused hearts. However, the direct influence of this hormone on adult cardiac myocyte contractile function is not well understood. Our aim is to investigate whether 1alpha,25-dihydroxyvitamin D(3) acutely modulates contractile function via PKC activation in adult rat cardiac myocytes. Sarcomere shortening and re-lengthening were measured in electrically stimulated myocytes isolated from adult rat hearts, and the vitamin D(3) response (10(-10) to 10(-7) M) was compared to shortening observed under basal conditions. Maximum changes in sarcomere shortening and relaxation were observed with 10(-9) M 1alpha,25-dihydroxyvitamin D(3). This dose decreased peak shortening, and accelerated contraction and relaxation rates within 5 min of administration, and changes in the Ca(2+) transient contributed to the peak shortening and relaxation effects. The PKC inhibitor, bis-indolylmaleimide (500 nM) largely blocked the acute influence of the most potent dose (10(-9) M) on contractile function. While peak shortening and shortening rate returned to baseline within 30 min, there was a sustained acceleration of relaxation that continued over 60 min. Phosphorylation of the Ca(2+) regulatory proteins, phospholamban, and cardiac troponin I correlated with the accelerated relaxation observed in response to acute application of 1alpha,25-dihydroxyvitamin D(3). Accelerated relaxation continued to be observed after chronic addition of 1alpha,25-dihydroxyvitamin D(3) (e.g. 2 days), yet this sustained increase in relaxation was not associated with increased phosphorylation of phospholamban or troponin I. These results provide evidence that 1alpha,25-dihydroxyvitamin D(3) directly modulates adult myocyte contractile function, and protein kinase C plays an important signaling role in the acute response. Phosphorylation of key Ca(2+) regulatory proteins by this kinase contributes to the enhanced relaxation observed in response to acute, but not chronic calcitriol.     

Cardiac-specific elevations in thyroid hormone enhance contractility and prevent pressure overload-induced cardiac dysfunction

Thyroid hormone (TH) is critical for cardiac development and heart function. In heart disease, TH metabolism is abnormal, and many biochemical and functional alterations mirror hypothyroidism. Although TH therapy has been advocated for treating heart disease, a clear benefit of TH has yet to be established, possibly because of peripheral actions of TH. To assess the potential efficacy of TH in treating heart disease, type 2 deiodinase (D2), which converts the prohormone thyroxine to active triiodothyronine (T3), was expressed transiently in mouse hearts by using the tetracycline transactivator system. Increased cardiac D2 activity led to elevated cardiac T3 levels and to enhanced myocardial contractility, accompanied by increased Ca(2+) transients and sarcoplasmic reticulum (SR) Ca(2+) uptake. These phenotypic changes were associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) 2a expression as well as decreased Na(+)/Ca(2+) exchanger, beta-myosin heavy chain, and sarcolipin (SLN) expression. In pressure overload, targeted increases in D2 activity could not block hypertrophy but could completely prevent impaired contractility and SR Ca(2+) cycling as well as altered expression patterns of SERCA2a, SLN, and other markers of pathological hypertrophy. Our results establish that elevated D2 activity in the heart increases T3 levels and enhances cardiac contractile function while preventing deterioration of cardiac function and altered gene expression after pressure overload.     

Identification of the t-type calcium channel (Ca(v)3.1d) in developing mouse heart

During cardiac development, there is a reciprocal relationship between cardiac morphogenesis and force production (contractility). In the early embryonic myocardium, the sarcoplasmic reticulum is poorly developed, and plasma membrane calcium (Ca(2+)) channels are critical for maintaining both contractility and excitability. In the present study, we identified the Ca(V)3.1d mRNA expressed in embryonic day 14 (E14) mouse heart. Ca(V)3.1d is a splice variant of the alpha1G, T-type Ca(2+) channel. Immunohistochemical localization showed expression of alpha1G Ca(2+) channels in E14 myocardium, and staining of isolated ventricular myocytes revealed membrane localization of the alpha1G channels. Dihydropyridine-resistant inward Ba(2+) or Ca(2+) currents were present in all fetal ventricular myocytes tested. Regardless of charge carrier, inward current inactivated with sustained depolarization and mirrored steady-state inactivation voltage dependence of the alpha1G channel expressed in human embryonic kidney-293 cells. Ni(2+) blockade discriminates among T-type Ca(2+) channel isoforms and is a relatively selective blocker of T-type channels over other cardiac plasma membrane Ca(2+) handling proteins. We demonstrate that 100 micromol/L Ni(2+) partially blocked alpha1G currents under physiological external Ca(2+). We conclude that alpha1G T-type Ca(2+) channels are functional in midgestational fetal myocardium.     

Phospholamban gene ablation improves calcium transients but not cardiac function in a heart failure model

Decreased amplitude and slower kinetics of cardiomyocyte intracellular calcium (Ca(i)(2+)) transients may underlie the diminished cardiac function observed in heart failure. These alterations occur in humans and animals with heart failure, including the TNF1.6 mouse model, in which heart failure arises from cardiac-specific overexpression of tumor necrosis factor alpha (TNF alpha). OBJECTIVE: Since ablation of phospholamban expression (PLBKO) removes inhibition of the sarcoplasmic reticulum (SR) Ca(2+) pump, enhances SR Ca(2+) uptake and increases contractility, we assessed whether ablation of phospholamban expression could improve cardiac function, limit remodeling, and improve survival in the TNF1.6 model of heart failure. METHODS: We bred PLBKO with TNF1.6 mice and characterized the progeny for survival, cardiac function (echocardiography), cardiac remodeling (hypertrophy, dilation, fibrosis), and Ca(2+)(i) transients and contractile function of isolated cardiomyocytes. RESULTS: PLB ablation did not improve survival, cardiac function, or limit cardiac chamber dilation and hypertrophy in TNF1.6 mice (TKO mice). However, contractile function and Ca(2+)(i) transients (amplitude and kinetics) of isolated TKO cardiomyocytes were markedly enhanced. This discordance between unimproved cardiac function, and enhanced Ca(2+)(i) cycling and cardiomyocyte contractile parameters may arise from a continued overexpression of collagen and decreased expression of gap junction proteins (connexin 43) in response to chronic TNF alpha stimulation. CONCLUSIONS: Enhancement of intrinsic cardiomyocyte Ca(2+)(i) cycling and contractile function may not be sufficient to overcome several parallel pathophysiologic processes present in the failing heart.