Periodic Reporting for period 1 - NO-STRESS (Nitric oxide regulation of repolarisation in the heart: role of mechanical stress)
Reporting period: 2018-09-30 to 2020-09-29
The muscle cells of the heart are called cardiomyocytes. When the heart beats, all the cardiomyocytes contract at the same time, leading to smooth movement of blood through the heart. Cardiomyocyte contraction occurs when electrical impulses trigger an electrical signal in the cardiomyocyte called an action potential. The action potential is the signal to make the cell contract, and is generated by tiny electrical currents flowing through specialised proteins called ion channels. Ion channels open in a specific sequence, and alterations in this sequence can lead to perturbations in the action potential, which can disrupt the synchronous contraction of cardiomyocytes. This is called fibrillation (irregular heart beat). Atrial fibrillation (AF) is the most common arrhythmia, and is a leading risk factor for stroke when blood clots forming in the atria travel to the brain. At the atrial cardiomyocyte level, changes in atrial electrical activity as a result of differences in the expression and function of ion channels are hallmark features of AF. The ion channel Kv1.5 contributes to the length of the action potential (AP), and as such, directly affects action potential duration (APD). Changes in APD occur early in the pathology of AF. Because of its atrial-specific localisation , Kv1.5 may be a target for AF therapies. Another feature of AF is a decrease in the enzyme neuronal nitric oxide synthetase, which produces nitric oxide (NO). There is growing evidence that NO can alter the activity of Kv1.5. This project was conducted to investigate how this regulation occurs in human cardiomyocytes, and whether this is altered in AF, a condition in which NO is depleted. In addition, mechanical regulation of cardiomyocyte electrical activity is receiving increasing attention as contributing factor in various cardiac pathologies where the mechanical environment is altered (including atrial fibrillation). As NO has been shown to be increased by mechanical stress, we wished to investigate whether mechanical regulation of Kv1.5 is altered by NO, as well as investigating the role of the recently characterised mechanically gated ion channel Piezo-1.
Increased knowledge in these areas will contribute to our understanding of AF, and may highlight new therapeutic targets.
The specific objectives were:
i) To investigate the mechanisms underlying the regulation of Kv1.5 by nNOS in human atrial myocytes from patients with and without AF.
ii) To understand how mechanical stress is linked to NO production and altered repolarisation in human physiology and AF.
iii) Investigate the role of the mechanically activated ion channel Piezo-1 in atrial cardiomyocytes
Increased knowledge in these areas will contribute to our understanding of AF, and may highlight new therapeutic targets.
The specific objectives were:
i) To investigate the mechanisms underlying the regulation of Kv1.5 by nNOS in human atrial myocytes from patients with and without AF.
ii) To understand how mechanical stress is linked to NO production and altered repolarisation in human physiology and AF.
iii) Investigate the role of the mechanically activated ion channel Piezo-1 in atrial cardiomyocytes
i) To investigate the mechanisms underlying the regulation of Kv1.5 by nNOS in human atrial myocytes from patients in sinus rhythm (SR) and atrial fibrillation (AF).
To investigate this objective, recordings of Kv1.5 activity in the presence of nNOS inhibition, using the specific inhibitor SMTC were made. Further, analysis of the interaction of Kv1.5 and nNOS protein were carried out by co-immunoprecipitation and immunocytochemistry. Localisation of Kv1.5 at the cell surface in response to nNOS inhibition was measured in rat atrial cardiomyocytes by total internal fluorescence microscopy. Finally, the nitrosylation of Kv1.5 was assessed by biotin switch assay. Conclusions from this objective were:
• In human atrial cardiomyocytes, Kv1.5 is not regulated by nNOS
• Kv1.5 does not physically associate with nNOS in the cell
• Kv1.5 is nitrosylated, but this is not related to nNOS expression, and this is not affected by atrial fibrillation (in which nNOS levels are decreased).
• In rat cardiomyocytes, nNOS inhibition may cause internalisation of Kv1.5 although it is unclear whether this is the case in humans
ii) To understand how mechanical stress is linked to NO production and altered repolarisation in human physiology and AF. work performed: Measurements of Kv1.5 activity in human atrial cardiomyocytes were made in the presence of shear stress. Total internal fluorescence microscopy was used to assess Kv1.5 cell surface localisation in response to shear stress in the presence of nNOS inhibition. Nitric oxide (NO) production was measured in atrial myocytes in the presence of shear stress.
Conclusions:
• Human cardiomyocytes do not respond to shear stress in the same way as rat cardiomyocytes
• In rats, shear stress induces production of NO, and inhibition of this process may prevent the increase in Kv1.5 at the plasma membrane previously observed. Therefore, NO production may be involved in the recruitment of channels induced by shear stress.
iii) Investigate the role of the mechanically activated channel Piezo-1 in cardiomyocytes
Piezo-1 expression was measured in atrial homogenates from patients in SR and AF. Calcium handling was assessed in guinea pig atrial cardiomyocytes in response to the Piezo-1 activator ‘Yoda’. Atrial natriuretic peptide release from atrial myocytes was measured by ELISA in response to Yoda and mechanical stress. Conclusions were:
• Human atrial homogenates contain piezo1 protein, and this may be altered in AF
• Calcium handling in atrial cardiomyocytes is altered in response to activation of piezo1
• ANP secretion from atrial cardiomyocytes may be altered by piezo1 activation.
Additional experiments were performed to investigate the regulation of Kv1.5 by nitric oxide in human cardiomyocytes, and how this contributes to the APD gradient between human left and right atria. The objective was to evaluate the role of nNOS in maintaining the APD in human atrial cardiomyocytes from both the LA and the RA, and to assess the importance of chamber differences in APD for arrhythmia susceptibility and maintenance.
Conclusions: The APD at all stages of repolarization was longer in RA than in LA myocytes. The currents responsible for the longer APD in the RA as a larger ICaL and smaller Ito. RA myocytes exhibited a larger IKur and IK1. nNOS inhibition , selectively shortened the RA APD thereby abolishing the APD difference between atria. nNOS protein content was significantly higher in the RA, in line with the more pronounced effect of nNOS inhibition in this chamber. Inhibition of the ultrarapid delayed rectifier K+ current (IKur) reversed the effect of SMTC on RA APD and restored the APD gradient, identifying IKur as the RA specific current responsible for the SMTC effect. To investigate whether the effect of nNOS depletion on the RA:LA APD difference has implications for atrial fibrillation, we performed in silico simulations which indicated that loss of atrial APD gradient promotes arrhythmia maintenance by increasing dominant frequencies.
IN summary, we identified nNOS as a key factor for maintenance of the different APD in human LA and RA. When nNOS is depleted this difference is abolished. Computer modelling predicts that loss of the APD difference may contribute to arrhythmia maintenance by affecting dominant frequencies.
These results are currently being prepared for submission to a cardiovascular journal.
To investigate this objective, recordings of Kv1.5 activity in the presence of nNOS inhibition, using the specific inhibitor SMTC were made. Further, analysis of the interaction of Kv1.5 and nNOS protein were carried out by co-immunoprecipitation and immunocytochemistry. Localisation of Kv1.5 at the cell surface in response to nNOS inhibition was measured in rat atrial cardiomyocytes by total internal fluorescence microscopy. Finally, the nitrosylation of Kv1.5 was assessed by biotin switch assay. Conclusions from this objective were:
• In human atrial cardiomyocytes, Kv1.5 is not regulated by nNOS
• Kv1.5 does not physically associate with nNOS in the cell
• Kv1.5 is nitrosylated, but this is not related to nNOS expression, and this is not affected by atrial fibrillation (in which nNOS levels are decreased).
• In rat cardiomyocytes, nNOS inhibition may cause internalisation of Kv1.5 although it is unclear whether this is the case in humans
ii) To understand how mechanical stress is linked to NO production and altered repolarisation in human physiology and AF. work performed: Measurements of Kv1.5 activity in human atrial cardiomyocytes were made in the presence of shear stress. Total internal fluorescence microscopy was used to assess Kv1.5 cell surface localisation in response to shear stress in the presence of nNOS inhibition. Nitric oxide (NO) production was measured in atrial myocytes in the presence of shear stress.
Conclusions:
• Human cardiomyocytes do not respond to shear stress in the same way as rat cardiomyocytes
• In rats, shear stress induces production of NO, and inhibition of this process may prevent the increase in Kv1.5 at the plasma membrane previously observed. Therefore, NO production may be involved in the recruitment of channels induced by shear stress.
iii) Investigate the role of the mechanically activated channel Piezo-1 in cardiomyocytes
Piezo-1 expression was measured in atrial homogenates from patients in SR and AF. Calcium handling was assessed in guinea pig atrial cardiomyocytes in response to the Piezo-1 activator ‘Yoda’. Atrial natriuretic peptide release from atrial myocytes was measured by ELISA in response to Yoda and mechanical stress. Conclusions were:
• Human atrial homogenates contain piezo1 protein, and this may be altered in AF
• Calcium handling in atrial cardiomyocytes is altered in response to activation of piezo1
• ANP secretion from atrial cardiomyocytes may be altered by piezo1 activation.
Additional experiments were performed to investigate the regulation of Kv1.5 by nitric oxide in human cardiomyocytes, and how this contributes to the APD gradient between human left and right atria. The objective was to evaluate the role of nNOS in maintaining the APD in human atrial cardiomyocytes from both the LA and the RA, and to assess the importance of chamber differences in APD for arrhythmia susceptibility and maintenance.
Conclusions: The APD at all stages of repolarization was longer in RA than in LA myocytes. The currents responsible for the longer APD in the RA as a larger ICaL and smaller Ito. RA myocytes exhibited a larger IKur and IK1. nNOS inhibition , selectively shortened the RA APD thereby abolishing the APD difference between atria. nNOS protein content was significantly higher in the RA, in line with the more pronounced effect of nNOS inhibition in this chamber. Inhibition of the ultrarapid delayed rectifier K+ current (IKur) reversed the effect of SMTC on RA APD and restored the APD gradient, identifying IKur as the RA specific current responsible for the SMTC effect. To investigate whether the effect of nNOS depletion on the RA:LA APD difference has implications for atrial fibrillation, we performed in silico simulations which indicated that loss of atrial APD gradient promotes arrhythmia maintenance by increasing dominant frequencies.
IN summary, we identified nNOS as a key factor for maintenance of the different APD in human LA and RA. When nNOS is depleted this difference is abolished. Computer modelling predicts that loss of the APD difference may contribute to arrhythmia maintenance by affecting dominant frequencies.
These results are currently being prepared for submission to a cardiovascular journal.
This project has reinforced the importance of nitric oxide for cardiac repolarisation in humans. A more complete understanding of the mechanisms by which nitric oxide affects ion channel activity in humans in both physiology and pathophysiology may contribute to targeted therapeutics for conditions in which nitric oxide production is impaired.