Nesfatin-1 is an eighty two amino acid long peptide cleaved from the N-terminal of its precursor protein, nucleobindin-2 (NUCB2). In addition to its metabolic actions, nesfatin-1 is also involved in modulating cardiovascular functions in rodents. Intracereberoventricular injection of nesfatin-1 increased mean arterial pressure in rats. In rats, nesfatin-1 acts as a post-conditioning agent and elicits cardioprotection against ischemia-reperfusion injury. It also affects the contraction and relaxation of the heart in rats in a dose dependent manner. Nesfatin-1 is emerging as a regulator of cardiovascular functions in rodents. However, whether nesfatin-1 regulates the cardiovascular system of non-mammals remain unknown. We hypothesized that nesfatin-1 is a modulator of cardiovascular functions in zebrafish. Here we characterized endogenous nesfatin-1 in zebrafish heart, and its effects on zebrafish cardiovascular physiology. We found that zebrafish cardiomyocytes express NUCB2 mRNA and nesfatin-1-like immunoreactivity. While NUCB2 mRNA was lower in unfed fish at 1 hour post-regular feeding time compared to the fish at 0 hour time point, it was observed that chronic food deprivation did not alter NUCB2 mRNA expression in zebrafish heart. Ultrasound imaging of zebrafish heart at 15 minutes post-intraperitoneal injection of nesfatin-1 (50 ng/g, 250 ng/g and 500 ng/g body weight) showed a dose-dependent inhibition of end-diastolic volume, but not end-systolic volume, while a significant increase in end-diastolic volume was found at the lowest dosage. However, these combined effects did not alter the stroke volume. A dose dependent decrease in heart rate and cardiac output was observed in zebrafish that received nesfatin-1. Nesfatin-1 caused a significant increase in the expression of Atp2a2a mRNA encoding the calcium-handling pump, SERCA2a, while it has no effects on the expression of calcium handling protein RyR1b encoding mRNA. NUCB2 mRNA and NUCB2/nesfatin-1 like immunoreactivity was detected in the cytoplasm of mouse HL-1 cardiomyocytes. High glucose increased NUCB2 mRNA expression in HL-1 cells. Genes involved in apoptosis, including Akt1, Caspases 1, 2, 3, and TNF were upregulated in the presence of 10 nM nesfatin-1. We also observed that NUCB2 mRNA expression was significantly increased in C57BL/6 mice heart in the presence of high glucose, whereas in diet induced obese C57BL/6 mice, NUCB2 mRNA expression was not altered. Together, our data supports the hypothesis that nesfatin-1 is expressed in the cardiovascular system of mouse and fish, and that nesfatin-1 modulates cardiovascular physiology in zebrafish.

It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10μM) and flufenamic acid (10 and 100μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca2+-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10μM) and flufenamic acid (10 and 100μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca2+-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

Electrical potentials exist across the membranes of nearly every cell type in the body. In addition, excitable cells, such as neurons, myocytes and even some endocrine cells elicit electrochemical fluctuations, action potentials (AP), in the cell membrane to initiate cell-to-cell communication or intracellular processes. The basis for the electrical potential is rooted within an array of complex interactions between monovalent ions and their associated membrane channels and transporters that regulate the flux of these charged species across the hydrophobic bilayer. Here, an expansion of our recently published work [1] will serve to explore the modern concepts regarding the origin of the AP as well as to examine the mechanisms by which intracellular calcium ([Ca2+]i) is regulated within the HL-1 mouse cardiac myocyte.

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca 2+]i, Ca2+ transients and membrane Ca2+ current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (120 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca 2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 28 nM); ? (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca 2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca 2+ transients. Triciribine (120 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca 2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca 2+]i required for excitation-contraction coupling in cardiomyoctyes.