Influx In Myocytes Research Articles

Scn1b expression in the adult mouse heart modulates Na+ influx in myocytes and reveals a mechanistic link between Na+ entry and diastolic function.

Voltage-gated sodium channels (VGSCs) are macromolecular assemblies composed of a number of proteins regulating channel conductance and properties. VGSCs generate Na+ current (INa) in myocytes and play fundamental roles in excitability and impulse conduction in the heart. Moreover, VGSCs condition mechanical properties of the myocardium, a process that appears to involve the late component of INa. Variants in the gene SCN1B, encoding the VGSC β1- and β1B-subunits, result in inherited neurological disorders and cardiac arrhythmias. But the precise contributions of β1/β1B-subunits and VGSC integrity to the overall function of the adult heart remain to be clarified. For this purpose, adult mice with cardiac-restricted, inducible deletion of Scn1b (conditional knockout, cKO) were studied. Myocytes from cKO mice had increased densities of fast (+20%)- and slow (+140%)-inactivating components of INa, with respect to control cells. By echocardiography and invasive hemodynamics, systolic function was preserved in cKO mice, but diastolic properties and ventricular compliance were compromised, with respect to control animals. Importantly, inhibition of late INa with GS967 normalized left ventricular filling pattern and isovolumic relaxation time in cKO mice. At the cellular level, cKO myocytes presented delayed kinetics of Ca2+ transients and cell mechanics, defects that were corrected by inhibition of INa. Collectively, these results document that VGSC β1/β1B-subunits modulate electrical and mechanical function of the heart by regulating, at least in part, Na+ influx in cardiomyocytes.NEW & NOTEWORTHY We have investigated the consequences of deletion of Scn1b, the gene encoding voltage-gated sodium channel β1-subunits, on myocyte and cardiac function. Our findings support the notion that Scn1b expression controls properties of Na+ influx and Ca2+ cycling in cardiomyocytes affecting the modality of cell contraction and relaxation. These effects at the cellular level condition electrical recovery and diastolic function in vivo, substantiating the multifunctional role of β1-subunits in the physiology of the heart.

Abstract 461: Sodium Influx Modulates the Modality of Cardiac Relaxation

Aging in experimental animals is coupled with protracted electrical recovery of the heart and increased late Na + current (I NaL ) in cardiomyocytes. These electrophysiological alterations are coupled with impaired cardiac relaxation, raising the possibility of a causative link between enhanced I NaL and diastolic dysfunction. To test this hypothesis, genetic and pharmacological interventions were introduced to assess the consequences of enhanced Na + influx in myocytes on diastolic properties of the mouse heart, together with effects on mechanical properties of isolated cells. Using Langendorff preparations, acute enhancement of I NaL with anemone toxin II increased diastolic and systolic pressure in the mouse heart. Importantly, a shift of the diastolic pressure-volume relationship toward higher pressure values was observed with activation of I NaL . To test the in vivo effects of increased Na + influx, mice with inducible, cardiac restricted deletion of the beta1 subunit of the Na + channel (Scn1b-KO) were employed. Scn1b-KO male mice presented protracted electrical recovery with respect to control (Ctrl) animals, a condition that was reversed by administration of a specific I NaL inhibitor (GS967, 0.5 mg/kg body weight). By invasive hemodynamics, left ventricular (LV) developed pressure was preserved in Scn1b-KO mice, but maximal velocities of pressure development and decay were attenuated by 16% and 25%, respectively. By echocardiography, LV end-diastolic volume and ejection fraction (EF) were preserved in Scn1b-KO. In contrast, using Doppler modality, LV filling pattern was altered and isovolumic relaxation time was prolonged by ~30%. I NaL inhibition (GS967) in Scn1b-KO mice ameliorated LV filling and normalized isovolumic relaxation time, without effects on EF. Using isolated cardiomyocyte preparations, Scn1b deletion had no consequences on fractional cell shortening, but led to a ~5% prolongation of kinetics of contraction and relaxation. Inhibition of I NaL (300 nM GS697) in Scn1b-KO myocytes accelerated contraction and relaxation kinetics and attenuated fractional shortening. In conclusion the late Na + current modulates the modality of myocyte contraction and relaxation with important effects on diastolic function of the heart.

Abstract 13874: Elevated [Na + ] i in Cardiac Myocytes from Diabetic Rats Due to Enhanced Na + - Glucose Cotransport

Intracellular Na + concentration ([Na + ] i ) is a key regulator of cardiac Ca 2+ cycling, contractility and metabolism. [Na + ] i is elevated in myocytes from failing hearts, leading to arrhythmias and oxidative stress. We hypothesized that myocyte [Na + ] i is also increased in type-2 diabetes (T2D) due to enhanced activity of the Na + -glucose cotransporter (SGLT). To test this hypothesis, we used myocardial tissue from humans with T2D and an animal model of late-onset T2D (HIP rats). We found increased SGLT expression in failing hearts from patients with T2D compared to non-diabetic individuals (by 55±16%) and in HIP rat hearts ( vs . age-matched wild-type, WT, littermates; by 59±17%). [Na + ] i , measured with the fluorescent indicator SBFI, is increased in myocytes from diabetic HIP rats, both at rest (14.7±0.9 mM compared to 11.4±0.7 mM in WT) and during electrical stimulation at 2 Hz (17.3±0.8 mM vs . 15.0±0.7 mM in WT). However, the Na + /K + -pump function (measured as the rate of pump-mediated [Na + ] i decline in intact myocytes) is not significantly altered in diabetic HIP rats. This result suggests that higher [Na + ] i is due to an increased Na + entry in HIP rat myocytes. Indeed, Na + influx, assessed as the rate of [Na + ] i rise upon Na + /K + -pump inhibition with 10 mM ouabain, was significantly larger in myocytes from diabetic HIP vs . WT rats (1.74±0.13 mM/min vs . 1.27±0.07 mM/min). SGLT inhibition with 250 μM phlorizin significantly reduced Na + influx in myocytes from diabetic HIP rats (to 1.08±0.20 mM/min), while it had no effect in the WT (1.14±0.21 mM/min). Phlorizin also significantly decreased glucose uptake in HIP rat myocytes (by 50±10 %) but not in WT, indicating an increased reliance on SGLT for glucose uptake in T2D hearts. In agreement with this result, the insulin-sensitive glucose uptake was greatly reduced in HIP rat myocytes vs . WT. These data suggest that SGLT is upregulated in diabetic hearts to compensate for reduced insulin-mediated glucose uptake. In summary, we found that [Na + ] i is elevated in myocytes from diabetic HIP rats due to an increased Na + entry via the Na + -glucose cotransporter. Higher [Na + ] i may contribute to arrhythmogenesis and oxidative stress in diabetic hearts.

Activation of reverse Na+-Ca2+exchange by the Na+current augments the cardiac Ca2+transient: evidence from NCX knockout mice

The hypothesis that Na(+) influx during the action potential (AP) activates reverse Na(+)-Ca(2+) exchange (NCX) and subsequent entry of trigger Ca(2+) is controversial. We tested this hypothesis by monitoring intracellular Ca(2+) before and after selective inactivation of I(Na) prior to a simulated action potential in patch-clamped ventricular myocytes isolated from adult wild-type (WT) and NCX knockout (KO) mice. First, we inactivated I(Na) using a ramp prepulse to 45 mV. In WT cells, inactivation of I(Na) decreased the Ca(2+) transient amplitude by 51.1 +/- 4.6% (P < 0.001, n = 14) and reduced its maximum release flux by 53.0 +/- 4.6% (P < 0.001, n = 14). There was no effect on diastolic Ca(2+). In striking contrast, Ca(2+) transients in NCX KO cardiomyocytes were unaffected by the presence or absence of I(Na) (n = 8). We obtained similar results when measuring trigger Ca(2+) influx in myocytes with depleted sarcoplasmic reticulum. In WT cells, inactivation of I(Na) decreased trigger Ca(2+) influx by 37.8 +/- 6% and maximum rate of flux by 30.6 +/- 7.7% at 2.5 mm external Ca(2+) (P < 0.001 and P < 0.05, n = 9). This effect was again absent in the KO cells (n = 8). Second, exposure to 10 mum tetrodotoxin to block I(Na) also reduced the Ca(2+) transients in WT myocytes but not in NCX KO myocytes. We conclude that I(Na) and reverse NCX modulate Ca(2+) release in murine WT cardiomyocytes by augmenting the pool of Ca(2+) that triggers ryanodine receptors. This is an important mechanism for regulation of Ca(2+) release and contractility in murine heart.

Mechanical stimulation of C2C12 cells increases m‐calpain expression, focal adhesion plaque protein degradation

Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. During embryonic development, myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load-induced signaling is now beginning to be understood as a factor which affects gene sequences, protein synthesis and an increase in Ca2+ influx in myocytes. Evidence of the involvement of Ca2+ -dependent activity in myoblast fusion, cell membrane and cytoskeleton component reorganization due to the activity of the ubiquitous proteolytic enzymes, calpains, has been reported. Whether there is a link between stretch- or load-induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have demonstrated that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown of specific focal adhesion proteins previously identified as substrates for this enzyme. We show that stimulation also leads to an increase in calpain activity in these cells. These data support the pivotal role for m-calpain in the control of muscle precursor cell differentiation and thus strengthen the idea of its implication during the initial events of muscle development.

Effects of calcitriol and its analogues, calcipotriol (MC 903) and 20-Epi-1α,25-dihydroxyvitamin D 3 (MC 1288), on calcium influx and DNA synthesis in cultured muscle cells

The fast actions of the secosteroid hormone 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3; calcitriolJ and the synthetic analogues calcipotriol (MC 903) and 20-epi-1α,25(OH) 2D 3 (MC 1288) on cell calcium influx were compared in rat duodenum enterocytes as well as in cells from chick embryo skeletal muscle (myoblasts) and heart (myocytes), at various concentrations (10 −12 to 10 −8 M) and treatment intervals (1–10 min). In enterocytes, at a concentration of 10 −11 M, MC 1288 was significantly more active than 1,25(OH) 2D 3 in rapidly stimulating 45Ca 2+ uptake by enterocytes (80 vs 38% above controls, respectively), whereas MC 903 was devoid of activity. However, calcipotriol increased Ca 2+ influx in myocytes and myoblasts to a greater extent than the natural hormone, whereas MC 1288 was more active only in myoblasts. Analogously to 1,25(OH) 2D 3, the fast MC 903- and MC 1288-induced stimulation of 45Ca 2+ uptake in enterocytes and muscle cells could be blocked by both verapamil and nifedipine. In addition, MC 903 and MC 1288 were more effective than 1,25(OH) 2D 3 in stimulating DNA synthesis in proliferating myoblasts and in inhibiting DNA synthesis in differentiating myoblasts. The results suggest, therefore, that modifications in the side-chain of the 1,25(OH) 2D 3 molecule increase its ability to modulate muscle cell Ca 2+ metabolism and growth. These findings are potentially relevant for the development of analogues for the treatment of vitamin D-dependent myopathies.