ALAMANDINE-(1–5) REDUCES L-TYPE CA²⁺ CURRENT IN MOUSE HEART CELLS

Background: Alamandine-(1–5) (ALA(1–5)) is a recently identified pentapeptide generated by angiotensinconverting enzyme through alamandine hydrolysis, placing it within the noncanonical renin–angiotensin system (Santos et al., 2026). Although ALA(1–5) has been shown to modulate cardiac contractility, the cellular mechanism underlying this effect has remained unknown. We investigated whether ALA(1–5) regulates ion channel activity in ventricular cardiomyocytes.
Methods: Adult mouse left ventricular cardiomyocytes were enzymatically isolated and maintained in Tyrode solution. Wholecell patchclamp recordings were performed at 22–25 °C using an EPC10 amplifier. Ltype Ca²⁺ currents (I) were elicited by 300ms depolarizing steps to 0 mV from a holding potential of –80 mV. ALA(1–5) was applied at 100 or 300 nM; A779 (1 µM) was used to assess MAS receptor involvement. Statistical significance was set at p < 0.05. CEUA #313/2024.
Results: ALA(1–5) produced a concentrationdependent inhibition of ICa,L. ALA(1–5) at 100 nM reduced current amplitude from –1.7 ± 0.21 nA to –1.1 ± 0.08 nA (35.3% inhibition, n = 6). Increasing ALA(1–5) to 300 nM decreased current from –2.5 ± 0.2 nA to –1.23 ± 0.07 nA (~51% inhibition, n=5). Preincubation for 10 min with A779 markedly attenuated ALA(1–5)–induced inhibition (300 nM; ~16%, n=4).
Conclusion: Our findings reveal that ALA(1–5) suppresses Ltype Ca²⁺ channel activity in cardiomyocytes, uncovering a previously unrecognized electrophysiological mechanism for this peptide. A779 partially attenuated the inhibitory effect, indicating that Masdependent signaling may modulate the response to ALA(1–5). These results redefine the functional role of ALA(1–5) within the noncanonical renin–angiotensin system and provide a mechanistic basis for its impact on cardiac contractility.