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Chad D Touchberry,
Ian K Bales,
Jessica K Stone,
Travis J Rohrberg,
Nikhil K Parelkar,
Tien Nguyen,
Oscar Fuentes,
Xia Liu,
Cheng-Kui Qu,
Jon J Andresen, Héctor H Valdivia,
Marco Brotto,
Michael J Wacker
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ABSTRACT: Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is the most recently identified phosphoinositide, and its functions have yet to be fully elucidated. Recently, members of our muscle group have shown that PI(3,5)P2 plays an important role in skeletal muscle function by altering Ca(2+) homeostasis. Therefore, we hypothesized that PI(3,5)P2 may also modulate cardiac muscle contractility by altering intracellular Ca(2+) ([Ca(2+)](i)) in cardiac myocytes. We first confirmed that PI(3,5)P2 was present and increased by insulin treatment of cardiomyocytes via immunohistochemistry. To examine the acute effects of PI(3,5)P2 treatment, electrically paced left ventricular muscle strips were incubated with PI(3,5)P2. Treatment with PI(3,5)P2 increased the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. These enhanced contractile responses were also observed in MIP/Mtmr14(-/-) mouse hearts, which we found to have elevated levels of PI(3,5)P2. In cardiac myocytes loaded with fura-2, PI(3,5)P2 produced a robust elevation in [Ca(2+)](i). The PI(3,5)P2-induced elevation of [Ca(2+)](i) was not present in conditions free of extracellular Ca(2+) and was completely blocked by ryanodine. We investigated whether the phosphoinositide acted directly with the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors; RyR2). PI(3,5)P2 increased [(3)H]ryanodine binding and increased the open probability (P(o)) of single RyR2 channels reconstituted in lipid bilayers. This strongly suggests that the phosphoinositide binds directly to the RyR2 channel. Thus, we provide inaugural evidence that PI(3,5)P2 is a powerful activator of sarcoplasmic reticulum Ca(2+) release and thereby modulates cardiac contractility.
Journal of Biological Chemistry 10/2010; 285(51):40312-21. · 4.77 Impact Factor
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ABSTRACT: Transient receptor potential melastatin (TRPM) is a subfamily of ion channels that are involved in sensing taste, ambient temperature, low pH, osmolarity, and chemical ligands. Melastatin 1/TRPM1, the founding member, was originally identified as melanoma metastasis suppressor based on its expression in normal pigment cells in the skin and the eye but not in aggressive, metastasis-competent melanomas. The role of TRPM1 and its regulation in normal melanocytes and in melanoma progression is not understood. Here, we studied the relationship of TRPM1 expression to growth and differentiation of human epidermal melanocytes. TRPM1 expression and intracellular Ca(2+) levels are significantly lower in rapidly proliferating melanocytes compared to the slow growing, differentiated melanocytes. We show that lentiviral short hairpin RNA (shRNA)-mediated knockdown of TRPM1 results in reduced intracellular Ca(2+) and decreased Ca(2+) uptake suggesting a role for TRPM1 in Ca(2+) homeostasis in melanocytes. TRPM1 knockdown also resulted in a decrease in tyrosinase activity and intracellular melanin pigment. Expression of the tumor suppressor p53 by transfection or induction of endogenous p53 by ultraviolet B radiation caused repression of TRPM1 expression accompanied by decrease in mobilization of intracellular Ca(2+) and uptake of extracellular Ca(2+). These data suggest a role for TRPM1-mediated Ca(2+) homeostasis, which is also regulated by ultraviolet B, in melanogenesis.
AJP Cell Physiology 08/2009; 297(3):C679-87. · 3.54 Impact Factor
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ABSTRACT: We describe the two-dimensional imaging of excitation-induced Ca gradients in isolated myocytes under physiological conditions, using a novel method of flash photography of fluo-3 fluorescence. This method is useful for showing the spatial distribution and reproducibility of rapidly triggered Ca release events, and their relationship to underlying structures. In normal rat myocytes, Ca sparks were evident 6ms after stimulation emerging from around t-tubules, as judged by co-localization with di-8-ANEPPS staining. Gaps in the spark pattern coincided with gaps in di-8-ANEPPS staining. Vacuolar fluo-3 uptake, previously identified as lysosomal, was prominent in some of the gaps, suggesting possible areas of t-tubule turnover. In normal dog myocytes, the beat-to-beat variance of Ca sparks was very low, t-tubular voids were small, and Ca gradients resolved rapidly. In myocytes from dogs with failure induced by rapid pacing, a reduced Ca transient was observed associated with increased areas that were void of sparks and t-tubules, and a greater beat-to-beat spark variance. These abnormalities resulted in a non-uniform spatial distribution of sparks, leading to Ca gradients across the cell that persisted for longer times after stimulation. Such Ca gradients could cause heterogeneous contraction and contribute to contractile failure.
Cell Calcium 03/2007; 41(2):123-34. · 3.77 Impact Factor
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Carlo G Tocchetti,
Wang Wang,
Jeffrey P Froehlich,
Sabine Huke,
Miguel A Aon,
Gerald M Wilson,
Giulietta Di Benedetto,
Brian O'Rourke,
Wei Dong Gao,
David A Wink,
John P Toscano,
Manuela Zaccolo,
Donald M Bers, Hector H Valdivia,
Heping Cheng,
David A Kass,
Nazareno Paolocci
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ABSTRACT: Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca(2+). A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca(2+) pump and the ryanodine receptor 2, leading to increased Ca(2+) uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca(2+)-release channels and accelerates Ca(2+) reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca(2+) transport. Contraction improves with no net rise in diastolic calcium. These changes are not induced by NO, are fully reversible by addition of reducing agents (redox sensitive), and independent of both cAMP/protein kinase A and cGMP/protein kinase G signaling. Rather, the data support HNO/thiolate interactions that enhance the activity of intracellular Ca(2+) cycling proteins. These findings suggest HNO donors are attractive candidates for the pharmacological treatment of heart failure.
Circulation Research 02/2007; 100(1):96-104. · 9.49 Impact Factor
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Circulation Research 10/2003; 93(6):487-90. · 9.49 Impact Factor
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ABSTRACT: The T-tubule membrane network is integrally involved in excitation-contraction coupling in ventricular myocytes. Ventricular myocytes from canine hearts with tachycardia-induced dilated cardiomyopathy exhibit a decrease in accessible T-tubules to the membrane-impermeant dye, di8-ANNEPs. The present study investigated the mechanism of loss of T-tubule staining and examined for changes in the subcellular distribution of membrane proteins essential for excitation-contraction coupling.
Isolated ventricular myocytes from canine hearts with and without tachycardia-induced heart failure were studied using fluorescence confocal microscopy and membrane fractionation techniques using a variety of markers specific for sarcolemmal and sarcoplasmic reticulum proteins.
Probes for surface glycoproteins, Na/K ATPase, Na/Ca exchanger and Ca(v)1.2 demonstrated a prominent but heterogeneous reduction in T-tubule labeling in both intact and permeabilised failing myocytes, indicating a true depletion of T-tubules and associated membrane proteins. Membrane fractionation studies showed reductions in L-type Ca(2+) channels and beta-adrenergic receptors but increased levels of Na/Ca exchanger protein in both surface sarcolemma and T-tubular sarcolemma-enriched fractions; however, the membrane fraction enriched in junctional complexes of sarcolemma and junctional sarcoplasmic reticulum demonstrated no significant changes in the density of any sarcolemmal protein or sarcoplasmic reticulum protein assayed.
Failing canine ventricular myocytes exhibit prominent depletion of T-tubules and changes in the density of a variety of proteins in both surface and T-tubular sarcolemma but with preservation of the protein composition of junctional complexes. This subcellular remodeling contributes to abnormal excitation-contraction coupling in heart failure.
Cardiovascular Research 08/2003; 59(1):67-77. · 6.06 Impact Factor
