Abstract
ABSTRACTThe functional expression of transient receptor potential cation channel of the ankyrin-1 subtype (TRPA1) has recently been identified in adult mouse cardiac tissue where stimulation of this ion channel leads to increases in adult mouse ventricular cardiomyocyte (CM) contractile function via a Ca2+-Calmodulin-dependent kinase (CaMKII) pathway. However, the extent to which TRPA1 induces nitric oxide (NO) production in CMs, and whether this signaling cascade mediates physiological or pathophysiological events in cardiac tissue remains elusive. Freshly isolated CMs from wild-type (WT) or TRPA1 knockout (TRPA1-/-) mouse hearts were treated with AITC (100 µM) and prepared for immunoblot, NO detection or ischemia protocols. Our findings demonstrate that TRPA1 stimulation with AITC results in phosphorylation of protein kinase B (Akt) and endothelial NOS (eNOS) concomitantly with NO production in a concentration- and time-dependent manner. Additionally, we found that TRPA1 induced increases in CM [Ca2+]i and contractility occur independently of Akt and eNOS activation mechanisms. Further analysis revealed that the presence and activation of TRPA1 promotes CM survival and viability following ischemic insult via a mechanism partially dependent upon eNOS. Therefore, activation of the TRPA1/Akt/eNOS pathway attenuates ischemia-induced CM cell death.
Highlights
The transient receptor potential channels of the ankyrin-1 subtype (TRPA1) are non-selective cation channels that are extensively expressed in sensory neurons [1,2,3] where they characteristically exhibit high permeability to calcium [4]
AktpS473 and eNOSpS1177, we determined that allyl isothiocyanate (AITC) elicits AktpS473 and eNOSpS1177 beginning at 2 min but most prominently at 5 min in WT CMs (183 ± 6.7% and 157 ± 8.0% of mean control value, respectively; Figure 1(a))
WT and TRPA1-/- CMs were loaded with an nitric oxide (NO) detection reagent and prepared for fluorescence microscopy in order to examine whether endothelial NOS (eNOS) phosphorylation occurs concomitantly with NO production
Summary
The transient receptor potential channels of the ankyrin-1 subtype (TRPA1) are non-selective cation channels that are extensively expressed in sensory neurons [1,2,3] where they characteristically exhibit high permeability to calcium [4]. Our lab previously identified that TRPA1 is expressed in murine cardiac muscle throughout the endocardium, myocardium and epicardium and colocalizes with TRPV1 at the costameres, z-discs and intercalated discs in the cardiomyocytes (CM) [8]. Our lab has recently demonstrated that TRPA1 stimulation of freshly isolated CMs enhances CM contractile function via a CaMKII-dependent pathway leading to increases in [Ca2+]i with a marked enhancement in the timing parameters of the Ca2+ transient and the CM contractile response [9]. A few downstream signaling mediators of TRPA1 in cardiac cells have been identified, the precise cellular signal transduction pathways and molecular mechanism(s) in which the ion channel is involved in modulating physiological or pathophysiological events in the heart remains to be fully elucidated
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