Introduction 7 11 1 10 6 + 12 8 6 2 Materials and methods All animal experimentation was carried out in accordance with the Animals (Scientific Procedures) Act 1986 and the local Institutional ethical review panel. Isolation of RNA Tris Real-time RT-PCR Real-time reverse transcription PCR (RT-PCR) was performed using TaqMan low-density arrays (Micro Fluidic Cards, Applied Biosystems, Foster City, CA). Each card consisted of 384 wells, preloaded with pre-designed fluorogenic TaqMan probes and primers, configured to allow detection of 48 transcripts for 7 experimental and 1 calibrator samples. Our calibrator sample consisted of a mixture of cDNA from EPI and ENDO regions. Each of the eight sample lanes in a card was loaded with 100 μl of a 1:1 mixture of cDNA (equivalent to 77 ng input RNA) and TaqMan Universial PCR master mix (Applied Biosystems). PCR was done according to the recommended protocol (50°C for 2 min and 94.5°C for 10 min, followed by 40 cycles at 97°C for 30 s and 59.7°C for 1 min) on an ABI RISM 7900HT Sequence detection system (Applied Biosystems). Data were collected with instrument spectral compensations by the Applied Biosystems SDS 2.2 software and analysed using the threshold cycle relative quantification method. Relative transcript expression was normalised to the housekeeper gene GAPDH. Mechanosensitive targets + 11 Isolation of left ventricular myocytes 2 3 2 4 4 2 2 2 Statistics t t P Results 1 P Table 1 Comparison of mRNA expression of mechanosensitive proteins from the EPI and ENDO region of rat left ventricle Assay ID reference Common name EPI ENDO t Mean ± SEM Mean ± SEM Slow response and/or hypertrophic  Agtr1a-Rn00578456_m1 Angiotensin II receptor 1A 1.64 ± 0.16 1.30 ± 0.22 NS  Edn1-Rn00561129_m1 Endothelin-1 1.17 ± 0.14 1.58 ± 0.24 NS  Ednra-Rn00561137_m1 Endothelin-1 receptor type A 1.19 ± 0.14 1.38 ± 0.12 NS   Ednrb-Rn00569139_m1 Endothelin-1 receptor type B 1.27 ± 0.17 1.93 ± 0.22 0.001 ENDO > EPI  Nppb-Rn00580641_m1 Brain natriuretic peptide 1.42 ± 0.15 2.77 ± 0.42 0.002 ENDO > EPI  Slc9a1-Rn00561924_m1 Sodium–hydrogen exchanger 1.33 ± 0.09 1.51 ± 0.17 NS Myofilaments  Myh6-Rn00568304_m1 α-myosin heavy chain 1.56 ± 0.12 1.37 ± 0.17 NS  Myh7-Rn00568328_m1 β-myosin heavy chain 1.20 ± 0.23 2.19 ± 0.36 0.006 ENDO > EPI Mechanosensitive channels  Kcnk2-Rn00597042_m1 Kcnk2 (TREK-1) 0.84 ± 0.04 1.04 ± 0.04 0.001 ENDO > EPI  Trpc1-Rn00585625_m1 Transient receptor potential canonical-1 0.72 ± 0.04 0.72 ± 0.05 NS Mechanotransducers (caveolae)  Cav-Rn00755834_m1 Caveolin-1 0.90 ± 0.03 0.96 ± 0.03 0.015 ENDO > EPI  Cav3-Rn0055343_m1 Caveolin-3 0.81 ± 0.03 0.85 ± 0.05 NS t P n NS n 1 1 Fig. 1 a SL b EPI ENDO P n n  Discussion This investigation of transcripts for proteins sensitive to stretch, or involved in mechanosensitive pathways, has allowed us to detect a number of small but nevertheless significant differences in mRNA levels between ENDO and EPI samples. Rapid and slow inotropic responses to axial stretch 10 2+ 50 3 2 9 MSCs and mechanotransducers 12 6 6 1 Although we found no evidence for a regionally different expression of caveolin-3, an essential component of cardiac caveolae, the role of caveolae may also be influenced by regional expression of the receptors and other signalling molecules that they aggregate. The observation that mRNA for caveolin-1 is greater in ENDO is intriguing as this isoform has only recently been identified in cardiac myocytes, and its role in myocyte caveolae is unknown. Indicators of cardiac hypertrophy 4 5 3 6 Assumptions and limitations 10 2 1 Conclusions 5 10 3 6