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.
Highlights
Isolation of Primary Cardiac Myocytes—To confirm the results demonstrated in our stable HL-1 cell line, we repeated essential experiments in adult primary cardiac myocytes
PI[3,5]P2 (0.5 M; n ϭ 5 animals) increased isometric force on average (1.61 Ϯ 0.23) when compared with vehicle (1.15 Ϯ 0.03) (Fig. 2B), whereas treatment of muscle strips with PI[4,5]P2 (0.5 M; n ϭ 5 animals) and PI[3]P (0.5 M; n ϭ 5 animals) did not increase isometric force development when compared with vehicle (1.36 Ϯ 0.14 and 1.25 Ϯ 0.17, respectively) (Fig. 2B)
1) PI[3,5]P2 is present and responsive to physiological stress in cardiac myocytes; 2) cardiac contractile force, rate of force development, and area were all increased following exposure to exogenous PI[3,5]P2 and in MIPϪ/Ϫ mice; 3) PI[3,5]P2 binds and increases the open probability of RyR2 channels; and 4) PI[3,5]P2 causes [Ca2ϩ]i release in cardiac myocytes by acting on RyR2-sensitive Ca2ϩ stores. These findings have led us to propose that PI[3,5]P2 induces increases in cardiac contractility by binding and opening RyR2, which increases the cytoplasmic Ca2ϩ levels needed for the ECC process and stimulation of CICR
Summary
The reduced skeletal muscle function of MIPϪ/Ϫ mice was attributed to the chronic leakage of Ca2ϩ from the sarcoplasmic reticulum due to direct binding of PI[3,5]P2 to the skeletal muscle ryanodine receptor (RyR1). These data strongly suggest that chronic PI[3,5]P2 dysregulation may be associated with dysfunctional Ca2ϩ handling and impaired contractile performance. PI[3,5]P2, Ca2؉ Homeostasis, and Cardiac Contractility tors) in a process known as Ca2ϩ-induced Ca2ϩ release (CICR) It is precisely this augmentation in Ca2ϩ release from the cardiac ryanodine receptors (RyR2) that allows for adjustments in cardiac contractility. We hypothesized that MIPϪ/Ϫ animals would demonstrate altered cardiac function
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