Cell volume control in phospholemman (PLM) knockout mice: do cardiac myocytes demonstrate a regulatory volume
James R Bell1, David Lloyd, Claire L Curl
1Cardiac Physiology, Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, London SE17EH, UK.
Abstract:
In addition to modulatory actions on Na+-K+-ATPase, phospholemman (PLM) has been proposed to play a role in cell volume regulation. Overexpression of PLM induces ionic conductances, with 'PLM channels' exhibiting selectivity for taurine. Osmotic challenge of host cells overexpressing PLM increases taurine efflux and augments the cellular regulatory volume decrease (RVD) response, though a link between PLM and cell volume regulation has not been studied in the heart. We recently reported a depressed cardiac contractile function in PLM knockout mice in vivo, which was exacerbated in crystalloid-perfused isolated hearts, indicating that these hearts were osmotically challenged. To address this, the present study investigated the role of PLM in osmoregulation in the heart. Isolated PLM wild-type and knockout hearts were perfused with a crystalloid buffer supplemented with mannitol in a bid to prevent perfusate-induced cell swelling and maintain function. Accordingly, and in contrast to wild-type control hearts, contractile function was improved in PLM knockout hearts with 30 mM mannitol. To investigate further, isolated PLM wild-type and knockout cardiomyocytes were subjected to increasing hyposmotic challenges. Initial validation studies showed the IonOptix video edge-detection system to be a simple and accurate 'real-time' method for tracking cell width as a marker of cell size. Myocytes swelled equally in both genotypes, indicating that PLM, when expressed at physiological levels in cardiomyocytes, is not essential to limit water accumulation in response to a hyposmotic challenge. Interestingly, freshly isolated adult cardiomyocytes consistently failed to mount RVDs in response to cell swelling, adding to conflicting reports in the literature. A proposed perturbation of the RVD response as a result of the cell isolation process was not restored, however, with short-term culture in either adult or neonatal cardiomyocytes.
Insights
Phospholemman (PLM) does not appear essential for cardiac cell volume regulation in response to osmotic stress. However, PLM knockout hearts showed improved function when protected from osmotic challenges, suggesting a role in cardiac osmoregulation.
Area of Science:
- Cardiovascular Physiology
- Cellular Biology
- Membrane Transport
Background:
- Phospholemman (PLM) modulates Na+-K+-ATPase and is implicated in cell volume regulation, with 'PLM channels' showing taurine selectivity.
- Overexpression of PLM in non-cardiac cells increases taurine efflux and augments regulatory volume decrease (RVD).
- Cardiac PLM's role in osmoregulation is unstudied, despite observed depressed cardiac function in PLM knockout mice under osmotic stress.
Purpose of the Study:
- To investigate the role of phospholemman (PLM) in cardiac osmoregulation.
- To determine if PLM is essential for limiting water accumulation and mediating RVD in cardiomyocytes.
- To assess the impact of PLM deficiency on cardiac function during osmotic challenges.
Main Methods:
- Isolated hearts from wild-type and PLM knockout mice were perfused with crystalloid buffer, with and without mannitol, to assess contractile function.
- Isolated adult cardiomyocytes from both genotypes were subjected to hyposmotic challenges.
- Cell width was tracked using the IonOptix video edge-detection system to measure cell swelling.
- Regulatory volume decrease (RVD) capacity was assessed in freshly isolated and cultured adult and neonatal cardiomyocytes.
Main Results:
- Contractile function was improved in PLM knockout hearts perfused with mannitol, indicating a protective effect against osmotic stress.
- Cardiomyocytes from both genotypes swelled equally under hyposmotic conditions, suggesting PLM is not essential for limiting water entry.
- Freshly isolated adult cardiomyocytes failed to exhibit RVD, and short-term culture did not restore this response in either genotype.
Conclusions:
- Phospholemman (PLM) is not critical for preventing water accumulation in cardiomyocytes during hyposmotic stress.
- PLM may play a role in maintaining cardiac function under osmotic challenge, potentially independent of direct control over water entry.
- The inability of isolated adult cardiomyocytes to perform RVD highlights challenges in studying cardiac osmoregulation in vitro.
