Introduction:

T-tubules (TTs) are invaginations of sarcolemma in cardiomyocytes important in excitation-contraction coupling. Mechanisms underlying the genesis and maintenance of TTs are poorly understood, but their loss in disease states contributes to the pathogenesis of heart failure and arrhythmia. Short-term culture of cardiac myocytes may provide a model system to study mechanisms underlying the maintenance of TT function.

 

Aims:

To examine changes in TTs and L-type calcium current (ICa) in adult rabbit ventricular myocytes (ARVMs) in short-term culture.

 

Method:

ARVMs were isolated from hearts of adult male rabbits and cultured on laminin-treated borosilicate glass coverslips for up to four days. TTs were identified by confocal imaging using Alexa-680-conjugated wheat germ agglutinin (WGA) and quantified by fast Fourier transform analysis (‘TT-power’). Junctophilin-2 (JPH2) staining was examined by immunofluorescence on the day of isolation (day 0) and following 4 days in culture (day 4). ICa was recorded from ARVMs at 37 °C using the whole-cell patch clamp technique on days 0,1,2 and 4. Response to 100 nM of the β-adrenoceptor agonist, isoprenaline (ISO), was examined in currents activated by depolarisation to 0 mV. Current densities were calculated as currents normalised to whole-cell capacitance, as an index of cell surface area. Data are reported as mean ± standard error and sample sizes as n cells/N animals. ICa density-voltage relations were plotted and fitted with Equation 1:

𝐼𝐶𝑎 = (G𝑚𝑎𝑥(Vm – V𝑟𝑒𝑣)/(1 + e^(Vm−Vhalf)/𝑘)),

where Gmax = maximal conductance density for ICa, Vm = command potential, Vrev = effective reversal potential for the current, Vhalf = voltage of half-maximal activation and k = slope factor. Fits to ICa density-voltage relations on days in culture were compared by extra sum-of-squares F test (GraphPad Prism 10.2). Responses to ISO were quantified as percentage increases relative to control and compared across days in culture by Welch’s one-way ANOVA). 

 

Results:

Cultured ARVMs became more rounded and whole-cell capacitance decreased exponentially with a time constant of 1.68 days (n/N = 16/3, 11/3, 18/5, 6/2 for days 0,1,2 and 4). TT-power showed a threefold-reduction from day 0 to day 4 (p = 0.001, unpaired t-test, n/N = 6/1 and 4/1). JPH2 staining intensity (AU) was significantly less at day 4 (25.5 ± 2.29; n/N = 5/1) compared to day 0 (36.1 ± 1.74; n/N = 6/1) of culture (p = 0.0045). At culture day 1, Gmax fell from 0.36 ± 0.03 nS/pF to 0.21 ± 0.02 nS/pF, but then recovered to 0.25 ± 0.02 nS/pF at day 2 and by day 4 had recovered to 0.33 ± 0.05 nS/pF (p < 0.0001, n/N = 11/3, 10/3, 13/5, 6/2 for days 0,1,2 and 4). The response to 100 nM ISO was preserved across days in culture (p = 0.91, n/N = 8/3, 6/3, 6/4, 3/2 for days 0,1,2 and 4). 

 

Conclusions:

Short-term culture of ARVMs was associated with loss of membrane surface area, loss of TT-power, reduced ICa density and reduced JPH2 staining, although responses to β-adrenoceptor stimulation were preserved. In future studies, we will use this experimental model to investigate mechanisms underlying loss of TTs and ICa.