Functional integrity of the T-tubular system in cardiomyocytes depends on p21-activated kinase 1

Abstract

p21-activated kinase (Pak1), a serine-threonine protein kinase, regulates cytoskeletal dynamics and cell motility. Recent experiments further demonstrate that loss of Pak1 results in exaggerated hypertrophic growth in response to pathophysiological stimuli. Calcium (Ca) signaling plays an important role in the regulation of transcription factors involved in hypertrophic remodeling. Here we aimed to determine the role of Pak1 in cardiac excitation-contraction coupling (ECC). Ca transients were recorded in isolated, ventricular myocytes (VMs) from WT and Pak1(-/-) mice. Pak1(-/-) Ca transients had a decreased amplitude, prolonged rise time and delayed recovery time. Di-8-ANNEPS staining revealed a decreased T-tubular density in Pak1(-/-) VMs that coincided with decreased cell capacitance and increased dis-synchrony of Ca induced Ca release (CICR) at individual release units. These changes were not observed in atrial myocytes of Pak1(-/-) mice where the T-tubular system is only sparsely developed. Experiments in cultured rabbit VMs supported a role of Pak1 in the maintenance of the T-tubular structure. T-tubular density in rabbit VMs significantly decreased within 24h of culture. This was accompanied by a decrease of the Ca transient amplitude and a prolongation of its rise time. However, overexpression of constitutively active Pak1 in VMs attenuated the structural remodeling as well as changes in ECC. The results provide significant support for a prominent role of Pak1 activity not only in the functional regulation of ECC but for the structural maintenance of the T-tubular system whose remodeling is an integral feature of hypertrophic remodeling.

Figure 1. Decreased Ca transient amplitude in Pak1^−/− VMs

F/F₀ plots from field stimulated WT (A) and Pak1^−/− (B) VMs. Ca transients in Pak1^−/− VMs had a decreased amplitude (C), prolonged recovery time to 90% decay (RT_90%)(D), and (E) Ca transient rise time, expressed as ‘time to peak’ (*, p < 0.05).

Figure 2. Ca influx and SR Ca load remain unchanged in Pak1^−/− VMs

(A) Current voltage relationship of I_Ca,L in WT and Pak1^−/− VMs obtained in the whole cell patch configuration from a holding potential of −50 mV (−40 to +40 mV), in 10 mV increments. (B) Representative current traces from WT and Pak1^−/− VMs during a voltage pulse to 10 mV. (C) I_Ca,L at −10 mV and (D) the amplitude of the caffeine (10 mmol/L) induced Ca transient were not significantly different in WT and Pak1^−/− VMs. (F) The propagation velocity of spontaneous Ca waves and their amplitude (E) was comparable in WT and Pak1^−/− VMs. (G) Western blotting analysis did not reveal differences in the protein expression of SERCA2a or _Caf1.2 in 4 and 6 month old Pak1^−/− VMs. α-actinin is shown as a loading control.

Figure 3. The AP in Pak1^−/− VMs exhibits a delayed repolarization

(A) The fractional release obtained from the ratio of the twitch vs. the caffeine induced transient amplitude, and (B) ECC gain obtained as the ratio of the twitch transient amplitude vs. I_Ca,L at a voltage pulse to −10 mV were reduced in Pak1^−/− VMs (*, p < 0.05). (C) Representative stimulation induced APs recorded from WT (black) and Pak1^−/−(grey) VMs. The APD at (C) 50% repolarization was significantly prolonged in Pak1^−/− VMs. (E) Suppression of I_to by 4-AP (100 μmol/L) increased the time to peak of the Ca transient in WT and Pak1^−/− VMs but maintained the difference between cell types (*, p < 0.05).

Figure 4. Pak1^−/− VMs exhibit changes in t-tubular density independent of hypertrophic remodeling

(A) Patch clamp recordings revealed a significantly reduced cell capacitance in Pak1^−/− VMs that did not correlate with a change in (B) VMs cell volume. Di-8-ANNEPS staining revealed a significant decrease in (C) t-tubular density within the WT and Pak1^−/− VMs. (D) Western blotting analysis did not reveal an increased expression of the hypertrophic marker proteins BNP and α-sk actin in 4 and 6 month old Pak1^−/− VMs. GAPDH and α-actinin are shown as loading controls. (E) Representative confocal images from a WT (top) and Pak1^−/− VM (bottom).

Figure 5. Pak1^−/− VMs exhibit an increased asynchrony in stimulation induced Ca release

Line scans and F/F₀ plots from (A) WT and (B) Pak1^−/− VMs obtained at a scanning frequency of 1200 lps. F/F₀ plots were obtained immediately before (black) and 20 ms after (red) stimulation of the Ca transient; time points are indicated by arrows. Pak1^−/− VMs displayed an increased number of regions (C) where [Ca] _i did not reach half maximal fluorescence (F50) 20 ms after the stimulus. Further an increased standard deviation of the time to peak (SD TT50; D) was recorded. Individual analysis of ‘early’ Ca release sites did not reveal differences in the time to peak (E) or ECC gain at −10 mV when only the sites of fast Ca rise were analyzed(*, p < 0.05).

Figure 6. Pak1 overexpression prevents t-tubular remodeling in rabbit VMs

Culture of adult ventricular myocytes after 24 h (black bars) results in a decrease of (A) the Ca transient amplitude and(B) a prolongation of the Ca transient rise time (time to peak) compared to Ctrl VMs at the time of isolation (white bars). In VMs transfected with constitutively active Pak1 (gray bars) culture induced changes in the Ca transient were attenuated. (C) T-tubular density in rabbit VMs non-transfected, or transfected with AdLacZ or AdPak1 after 0, 24, and 48 h of culture. T-tubular remodeling was attenuated in AdPak transfected cells(*, p < 0.05).(D) Western blot analysis of endogenous Pak1 (top panel) after 24h in culture indicated a loss of detectable Pak1 expression whereas adPak1 overexpression in VMs occurred at 24h and 48 h (bottom panel) of culture; α-actinin or GAPDH are shown as loading controls, respectively. Representative original confocal images of di-8-ANNEPS stained rabbit VMs that were (E) non-transfected or (F) transfected with AdPak1.

Figure 7. Changes in Pak1^−/− ECC are based on t-tubular remodeling

Representative original confocal images of di-8-ANNEPs stained atrial myocytes isolated from right atria of (A) WT and (B) Pak1^−/− mice. The staining did not reveal an organized t-tubular system. In contrast to VMs (C) Ca transient amplitude and (D) rise time (time to peak) were unchanged in WT and Pak1^−/− atrial myocytes.