Multiple H+ sensors mediate the extracellular acidification-induced [Ca2+]i elevation in cultured rat ventricular cardiomyocytes

Acidosis has been known to cause “Ca2+ transients”, however, the mechanism is still uncertain. Here, we demonstrated that multiple H+ sensors, such as ASICs, TRPV1 and proton-sensing G protein coupled receptors (GPCRs) are involved in extracellular acidification-induced intracellular calcium ([Ca2+]i) elevation. By using calcium imaging measures, we observed that both ASIC and TRPV1 channels inhibitors suppressed the [Ca2+]i elevation induced by extracellular acidosis in cultured rat cardiac myocytes. Then, both channels mRNA and proteins were identified by RT-PCR, western blotting and immunofluorescence. ASIC-like and TRPV1-like currents were induced by extracellular acidification, suggesting that functional ASIC and TRPV1 channels jointly mediated extracellular calcium entry. Furthermore, either pre-exhaustion of sarcoplasmic reticulum (SR) Ca2+ with thapsigargin or IP3 receptor blocker 2-APB or PLC inhibitor U73122 significantly attenuated the elevation of [Ca2+]i, indicating that the intracellular Ca2+ stores and the PLC-IP3 signaling also contributed to the acidosis-induced elevation of [Ca2+]i. By using genetic and pharmacological approaches, we identified that ovarian cancer G protein-coupled receptor 1 (OGR1) might be another main component in acidosis-induced release of [Ca2+]i. These results suggest that multiple H+-sensitive receptors are involved in “Ca2+ transients” induced by acidosis in the heart.

ments in cultured rat cardiac myocytes. As shown in Fig. 1a, a rapid and transient elevation of intracellular calcium ([Ca 2+ ] i ) was observed when the pH value of extracellular solution was rapidly lowered from 7.4 to 6.0 or 5.0. This elevation is reversible and could be stably induced when another acidosis solution was given again. The peak of Δ F/F was 1.37 ± 0.08 at pH 6.0 (n = 15 cells) and 2.93 ± 0.22 at pH 5.0 (n = 19 cells, P < 0.01 vs pH 6.0, Student's t-test, Fig. 1a). These results suggest that extracellular acidification (pH 6.0 or pH 5.0) can elevate the [Ca 2+ ] i in primary cultured cardiomyocytes with a pH-dependent manner. We then chose pH 5.0 in the following experiments.
In order to explore the possibility of the involvement of ASICs and TRPV1 in the influx of extracellular Ca 2+ , we used pharmacological inhibitors for ASICs and TRPV1. It was shown that, bath application of ASIC blockers amiloride (300 μ M, a nonspecific inhibitor of ASICs) inhibited the increase of [Ca 2+ ] i by 50.07 ± 5.70% (n = 12 cells, P < 0.01 vs control, Student's t-test, Fig. 1b), and PcTx1 (10 nM, a specific inhibitor of ASIC1) inhibited the increase by 53.51 ± 3.31% (n = 12 cells, P < 0.01 vs control, Student's t-test, Fig. 1c), while incubation of TRPV1 non-specific antagonist ruthenium red (10 μ M) inhibited [Ca 2+ ] i rise by 56.16 ± 6.22% (n = 16 cells, P < 0.01 vs control, Student's t-test, Fig. 1d). Then, we added amiloride (300 μ M) and ruthenium red (10 μ M) simultaneously and found that the Ca 2+ elevation induced by pH drop was largely blocked, which was decreased by 82.61 ± 4.18% (n = 10 cells, P < 0.01 vs control, Student's t-test, Fig. 1e). Since ruthenium red is also an effective blocker of ryanodine receptors, we chose CPZ, a specific inhibitor of TRPV1 to evaluate its effect on [Ca 2+ ] i elevation again. The inhibition ratio was 39.33 ± 5.91% with CPZ single treatment and it raised to 68.38 ± 2.03% when combining with PcTx1 (n = 17~23 cells, P < 0.01 vs control, ANOAN followed by LSD, Fig. 1f). These results indicate that both ASIC channels and TRPV1 channel are the main sources of [Ca 2+ ] i elevation in cultured rat cardiac myocytes.
Expressions of ASICs in cultured rat cardiac myocytes. In order to confirm whether ASIC channels are expressed on cardiac myocytes, specific antibodies were used for western blotting analysis. The results showed specific ASIC1, 2a and 3 bands in cultured rat cardiac myocytes, and the molecular weights of all ASIC subunits in rat heart cells were near 72 kDa. After adding peptides to react with respective antibodies, the corresponding bands almost disappeared ( Fig. 2a and Supplementary Figs S1a and S2). We further performed RT-PCR to clarify ASICs gene expressions from cultured rat ventricular myocytes, the products corresponding to ASIC1, 2 and ASIC3 were similar between cardiomyocytes and brain cortex, which were 140, 203, and 107 bp, respectively (Fig. 2b). These results indicate that three ASIC subunits transcripts and proteins are expressed in rat cardiomyocytes.
Then, we observed the distribution of different ASIC proteins in the heart tissues by immunofluorescence. As shown in Fig. 2c, ASIC proteins were recognized by corresponding antibodies and the double immunofluorescence staining showed different spread immunosignals among three subunits in the cultured rat cardiomyocytes. ASIC1, but not ASIC3 protein predominantly merged with nuclear marker Hoechst 33258, ASIC2a had a uniform distribution, not only in nuclear but also in cytoplasm. The various distributions of ASICs subunits suggested the distinct intracellular functions and characteristics of ASICs on the heart. Characteristics of ASIC-like currents in cardiac myocytes. The recorded currents were induced by extracellular acidosis in cultured rat cardiomyocytes. At a holding potential of − 80 mV, a rapid reduction of extracellular pH to 6.0 resulted in a fast activating ASIC-like inward current. These currents could be blocked by 100 μ M amiloride and 10 nM PcTx1, and the amplitude decreased by 71.33 ± 3.01% (n = 3 cells, P < 0.05 vs control, Student's t-test) and 46.67 ± 4.25% (n = 4 cells, P < 0.05 vs control, Student's t-test), respectively. The inhibitory effect was mostly recovered after washout (Fig. 3a), further confirming that these currents were ASIC-like currents.
In order to eliminate the influence of culture conditions and developmental stage, we also recorded ASIC-like currents in adult rat acute isolated ventricular cardiocytes. As shown in Fig. 3b, the amplitude of inward current was inhibited by 74.21 ± 3.34% (n = 3 cells, P < 0.05 vs control, Student's t-test) with 100 μ M amiloride, and recovered mostly after washout. Thus, ASIC currents in cardiomyocytes displayed similar electrophysiological property as that in nervous system. Local decrease in extracellular solution to pH 7.0 was sufficient to induce an ASIC-like current, and the amplitudes of ASIC current were increased gradually along with the decrease in pH from 7.4 to 7.0, 6.0, 5.0 and 4.0. Fitting with Hill equations, the curve of pH-current density was "S" shape, and the pH 50 (pH for half-maximal activation) was 5.73 ± 0.18 (n = 7~11 cells, Fig. 3c). These results indicate that ASIC currents in the rat cardiomyocytes are pH-dependent.
Expression and characteristic of TRPV1 channel in rat cardiac myocytes. Meanwhile, another H + -sensitive channel, TRPV1 channel was detected. Firstly, TRPV1 transcripts were shown in cultured cardiocytes of rat as well as in cortex, the products were at 282 bp (Fig. 4a). Then at the protein level, the single prominent bands of 55~72 kDa were recognized in cultured ventricular myocytes. The detected TRPV1 protein should be specific because adding homologus peptide led to negative consequence ( Fig. 4b and Supplementary Fig. S1b). Subsequently, the double immunostaining results showed that TRPV1 protein evenly expressed in rat ventricular myocytes. Using Hoechst 33258 as a nuclear indicator, we found that TRPV1 protein was distributed not only in the nucleus but also in the plasma of cardiac cells (Fig. 4c).
Then, we investigated the electrical properties of TRPV1 channel in cultured and acute isolated cardiomyocytes. TRPV1-like currents were recorded at a holding potential of − 80 mV by a rapid reduction of extracelluar pH from 7.4 to 6.0, and they were activated rapidly and sustained without desensitization until extracellular pH returned to 7.4. The currents in both kinds of cells could be reversibly blocked by capsazepine (CPZ, 20 μ M), a neuronal TRPV1-specific inhibitor (n = 3 cells, P < 0.05 vs control, Student's t-test, Fig. 4d), indicating that the sustained currents are actually mediated by TRPV1. Along with the pH value decreasing from 7.4 to 7.0, 6.0, 5.0, the current amplitude was increased gradually (Fig. 4e). In order to demonstrate the presence of TRPV1, capsaicin, a specific agonist of TRPV1 channel was used here. A robust elevation of [Ca 2+ ] i was seen by capsaicin (10 μ M, n = 16 cells) stimulation and CPZ (20 μ M) blocked it largely (n = 16 cells, P < 0.01 vs control, Student's t-test, Fig. 4f). Thus, other than ASICs, TRPV1 channel was another H + -sensitive channel in the heart.
To summarize the ratios, we found that after exposure to extracellular pH 6.0, 34.25% in cultured ventricular cells and 23.08% in acute-isolated ventricular cells displayed TRPV1 -like currents, while ASIC -like currents in these two kinds of cells took up to 43.83% and 61.54%, respectively, the remaining cells had no response to extracellular acidosis.

Phospholipase C (PLC) -inositol 1,4,5-trisphosphate (IP 3 ) signaling and sarcoplasmic reticulum (SR) mobilization mediate acidosis-induced Ca 2+ elevation in the absence of extracellular
Ca 2+ . Although ASIC channels and TRPV1 channel were the main sources of [Ca 2+ ] i elevation in cultured rat cardiac myocytes, both specific inhibitors could not block acidosis-induced Ca 2+ elevation completely, indicating that other mechanism should be involved. Here, when Ca 2+ was depleted by removing extracellular Ca 2+ and adding EGTA, we observed an interesting phenomenon. The elevation of [Ca 2+ ] i triggered by acidification in the absence of extracellular Ca 2+ was declined from 2.82 ± 0.20 (n = 25 cells) to 1.03 ± 0.12 (n = 19 cells, P < 0.01, Student's t-test, Fig. 5a), whose inhibition ratio was much close to the effects of PcTx1 and CPZ combination  Fig. 1f). This result suggests that ASIC and TRPV1 channels might be the component of extracellular Ca 2+ entry, and the left elevation of Ca 2+ might be derived from intracellular Ca 2+ stores. Since sarcoplasmic reticulum is the possible source of intracellular Ca 2+ elevation in cardiomyocytes, and thapsigargin (TG) is the inhibitor of SR Ca 2+ -ATPase that can exhaust the Ca 2+ content, so TG was used here to examine the role of SR. The results showed that the extracellular acidification-induced [Ca 2+ ] i elevation in the absence of extracellular Ca 2+ was almost totally abolished by TG (3 μ M) with the inhibition ratio of 90.59 ± 1.46% (n = 15 cells, P < 0.01 vs control n = 14 cells, Student's t-test, Fig. 5b), indicating that SR mobilization mediates acidosis-induced elevation of Ca 2+ that derived from intracellular Ca 2+ stores.
We then testify the contribution of SR in the extracellular acidification-triggered [Ca 2+ ] i elevation. Since GPCRs are reported to couple to PLC, liberating IP 3 to bind to IP 3 R, and IP 3 R is a kind of receptor that expressed in SR membrane, mediating Ca 2+ release 21 , the changes of [Ca 2+ ] i with 2-APB, the blocker of IP 3 receptor (IP 3 R) and U73122, the inhibitor of PLC in cytoplasm were measured here. As shown in Fig. 6a and b, treatment with 2-APB (200 μ M) and U73122 (3 μ M) significantly decreased the amplitude of Ca 2+ elevation evoked by extracellular acidification in the absence of extracellular Ca 2+ , the inhibitory ratios were 49.80 ± 4.85% (n = 15 cell) and 53.93 ± 5.14% (n = 13 cells, P < 0.01 vs control, Student's t-test), respectively. To rule out the influence of DMSO on responses of cardiomyocytes to acidification, we measured the acidic solution (pH 5.0)-triggered Ca 2+ signals with incubation of 0.1% DMSO, and no significant decay of Ca 2+ signals was observed after DMSO exposure (n = 18 cells, P > 0.05 vs control n = 12 cells, Student's t-test, Fig. 6c). Hence, these results suggested that the elevations of intracellular Ca 2+ triggered by extracellular acidification in the absence of extracellular Ca 2+ might be due to the activation of PLC/IP3 receptor and the following mobilization of SR.
The involvement of OGR1 in the extracellular acidification-induced Ca 2+ mobilization in rat ventricular cardiomyocytes. Considering that Gq protein has been accepted as the upstream signal of PLC activation, we asked how extracellular acidification could activate Gq protein. In previous studies, the family of proton sensing GPCRs have been reported to play a role in linking extracellular protons to Gq or Gs signals 19 . Thus, the gene expression of the four subtypes (TDAG8, GPR4, OGR1, and G2A) of GPCRs was examined in primary cultured rat ventricular cardiomyocytes. As shown in Fig. 7a, the specific DNA bands of TDAG8, GPR4, and OGR1 were detected in primary cultured cardiomyocytes, however, G2A product was absence in the same cardiomyocytes. Thus, the gene type of proton sensing G protein coupled receptors was clearly determined in primary cultured ventricular cardiomyocytes.
Among the already confirmed subtypes of G protein coupled receptors (TDAG8, GPR4, and OGR1) in rat ventricular cardiomyocytes, only OGR1 was considered to be the primary protein that transferred extracellular proton signals to Gq proteins 19 . Based on the PCR results, we further investigated the expression of OGR1 in in  vitro cardiomyocytes using specific OGR1 antibody. As shown in Fig. 7b (also see Supplementary Fig. S3) and 7c, the immunofluorescence and western blotting analyses clearly proved the existence of OGR1 in primary rat ventricular cardiomyocytes. It is reported that OGR1 has the maximal activation at pH 6.8, and pH 7.6 solution will make it more sensitive to pH change 22 , so we changed the extracelluar pH from 7.6 to 7.0 to investigate the OGR1 activation. The results showed a mild elevation of [Ca 2+ ] i in cultured myocardiac cells with the peak of Δ F/F 1.31 ± 0.09 (n = 15 cells), and Cu 2+ , the inhibitor of the protonation of extracellular histidines residues in OGR1, could inhibit this elevation to 0.46 ± 0.05 (n = 9 cells, P < 0.01 vs control, Student's t-test, Fig. 7e), further indicating the existing of OGR1. Next, to confirm the involvement of OGR1 in the extracellular acidification-triggered [Ca 2+ ] i elevation, we evaluated the effects of Cu 2+ in the absence of extracellular Ca 2+ . Our results showed that Cu 2+ markedly attenuated the elevation of [Ca 2+ ] i triggered by pH 5.0 acidic solutions with the inhibitory ratio of 47.91 ± 8.35% (n = 15 cells, P < 0.01 vs control n = 16 cells, Student's t-test, Fig. 7d). Thus, the sensor of extracellular protons in cardiomyocytes that triggered the elevation of [Ca 2+ ] i was OGR1. In order to verify both ASIC/ TRPV1 channels and OGR1 protein contribute to acidosis-induced Ca 2+ release, we combined three inhibitors of PcTx1, CPZ and Cu 2+ together, the results showed nearly complete blockade (84.35% ± 1.36%, n = 25 cells, P < 0.01 vs control n = 20 cells, Student's t-test) of Ca 2+ transient induced by pH 5.0 solution (Fig. 7f). Taken together, there are two components for Ca 2+ elevation in response to elevated external protons, one is ASIC/ TRPV1 channel and another is OGR1protein.

Discussion
In the present study, we first found that extracellular acidification can induce an elevation of [Ca 2+ ] i in cultured rat cardiac myocytes; We then identified that ASICs and TRPV1 were expressed in the cardiac myocytes and jointly mediated extracellular calcium entry; Thirdly, another sensor of extracellular protons OGR1 was also found in cardiomyocytes, it mediated the IP 3 R-gated mobilization of intracellular Ca 2+ in SR via OGR1-PLC-IP 3 -IP 3 R signaling pathway.
Acidosis is a detrimental condition accompanied with some cardiac disease, for example, myocardial ischemia. The outcome of acidosis is the increase of [Ca 2+ ] i followed by various functional changes in cardiomyocytes 1 . In the present study, application of extracellular buffer at pH 6.0 or 5.0 to primary cardiomyocytes also showed a robust elevation of [Ca 2+ ] i in a pH-dependent manner. Since H + -sensitive ion channels or receptors  may be one of the mediators for the influx of extracellular Ca 2+ , the effects of ASICs and TRPV1 were investigated. The antagonists of ASIC and TRPV1 channels inhibited in part the augmentation of [Ca 2+ ] i , respectively. Meanwhile, blockade of both ASIC and TRPV1 channels inhibited the elevation of [Ca 2+ ] i more significantly, and the inhibition ratio was similar to the effect of EGTA, where extracellular Ca 2+ was absent. Therefore, ASIC and TRPV1 channels contributed prominently to the acid-evoked Ca 2+ influx.
Although ASICs and TRPV1 are mainly found in the nervous system, both channels have also been reported to express in cardiovascular systems [16][17][18]23,24 , however, there is no evidence for their distribution in the heart. Here, we first demonstrated the presence of the mRNAs and proteins of ASIC1, 2 and 3 in the cultured myocardiac cells. Moreover, different from the predominance of ASICs in neuronal membranes, the localization of ASIC subunits in the cardiomyocytes had unique specificity. This variability resulted in disparate ASIC currents in the myocardium. Therefore, we calculated the pH 50 of ASIC currents in the myocardiocytes, the value of 5.7 was different from any subunits of ASICs in neurons. Since in the central nervous system, the pH 50 of homomeric ASIC1a channels is 6.2 or 6.8, ASIC1b 5.9, homomeric ASIC2a channels 4.4, while ASIC3 has a biphasic response with a fast desensitizing current followed by a sustained component (pH 50 : 6.7) 9 . As we known, different ASIC subunits form the heterotypic channels, the shape and characteristics including pH 50 of current would be changed correspondingly 25 . Thus, we presumed that the principal ASIC currents on the heart were produced by heterogenous ASIC channels. Other than ASIC channels, we also identified TRPV1 channel in the cardiomyocytes, which distributed evenly through cytoplasma and nucleus. The decrease of extracellular pH induced TRPV1-like currents in a pH-dependent manner. Similar with cultured neonatal rat cardiomyocytes, both ASIC and TRPV1 -like currents could be induced in acute isolated adult rat cardiomyocytes. The recording of currents in cardiac myocytes by different experimental conditions and developmental phases further confirmed that both cardiac pH-sensitive channels are functional.
In the present study, the depletion of extracellular Ca 2+ significantly attenuated the increase in [Ca 2+ ] i that triggered by extracellular acidification, suggesting the possible contributions of intracellular Ca 2+ stores in acidosis-induced elevation of [Ca 2+ ] i . Under absence of extracellular Ca 2+ conditions, when the Ca 2+ contents in SR were exhausted by using TG, the elevation of [Ca 2+ ] i was almost totally abolished, demonstrating that acidosis elevated [Ca 2+ ] i via prompting the release of Ca 2+ from SR in cardiomyocytes. Although it is well accepted that SR can be mobilized by ryanodine receptor activation 26 , IP 3 receptor has attracted more and more attentions recently, especially for their functions in cardiomyocytes. The activity of IP 3 receptors can be directly potentiated by intracellular IP 3 , triggering the release of Ca 2+ that elicits excitation-contraction coupling or cardiac hypertrophy, and predisposes ventricle to arrhythmia [27][28][29][30][31] . In our experiments, blockade of IP 3 receptor with 2-APB significantly attenuated the acidification-induced [Ca 2+ ] i elevation in cardiomyocytes, which was also observed in the specific inhibitor of PLC, U73122-treated groups, thus, IP 3 receptors should mediate the extracellular acidification-induced [Ca 2+ ] i elevation via IP 3 -PLC signaling. However, the possible contributions of RyRs to the [Ca 2+ ] i elevation could not be completely ruled out. In fact, there is a clear potential for cross-talking between RyRs and IP3R-mediated Ca 2+ signaling pathways in heart muscle 32 . Hence, the relative contributions of RyR-gated Ca 2+ stores to acidification-induced [Ca 2+ ] i elevation would be further investigated in cardiomyocytes.
The activation of IP 3 -generated PLC is mediated by Gq protein that are usually coupled with GPCRs 33,34 . Here, we asked whether the activation of PLC by extracellular acidification in cardiomyocytes could be resulted from the activation of some Gq-associated GPCRs. Fortunately, a group of proton sensing G protein coupled receptors provided us some important information. The family of proton sensing G protein coupled receptors contains four subtypes: TDAG8, GPR4, OGR1, and G2A. Among them, TDAG8 and GPR4 are mainly Gs-coupled and the latter two subtypes are usually Gq-coupled 19,22 . In light of the above characteristics, we investigated the gene expression of these four proteins in primary cultured cardiomyocytes. Interestingly, the level of TDAG8, GPR4 and OGR1 proteins were high, while G2A was absence in primary cultured cardiomyocytes, suggesting that OGR1 was the most potential Gq-associated GPCR for the intracellular Ca 2+ mobilization from SR by acidification. Using specific antibody for OGR1, we also examined the existence of OGR1 in ventricular cardiomyocytes, and calcium imaging found that OGR1 had a relative high sensitivity to protons, when extracellular pH was lowered from 7.6 to 7.0, it has already been activated. Furthermore, the possible involvement of OGR1 in the proton-induced activation of PLC and the following mobilization of intracellular Ca 2+ were also tested by application of Cu 2+ . Pre-incubation of Cu 2+ significantly attenuated the elevation of [Ca 2+ ] i via binding to essential histidine residues in OGR1 22 . Although the inhibitory effects of Cu 2+ was properly non-specific, the specificity of Cu 2+ for proton sensing GPCRs other than OGR1 has not been reported. Hence, we concluded that OGR1 mediated the IP 3 R-gated mobilization of intracellular Ca 2+ in SR via sensing extracellular protons.
In summary, the present study found that an extracellular acidification can induce"Ca 2+ transients" in cultured rat cardiac myocytes. As H + -sensitive ion channels, ASICs and TRPV1 can be activated by acidosis solution and mediate extracellular Ca 2+ entry. On the other hand, OGR1 mediates the mobilization of intracellular Ca 2+ from SR via OGR1-PLC-IP 3 -IP 3 R signaling pathway (Fig. 8). The results and conclusion from the present study will provide a new clue and evidence for understanding the possible roles and mechanisms of H + -sensitive receptors in heart ischemia.

Methods
In these studies, all experimental protocols were conducted in accordance with Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication, 8 th Edition, 2011) and approved by the Committee of Animal Care of Huazhong University of Science and Technology.
Culture of ventricular myocytes. Firstly, neonatal Sprague-Dawley rats (1-2 days old) were euthanized by decapitation, ventricular tissues were excised and washed three times in phosphate-buffered saline (PBS), and then were cut into shivers with the size less than 1 mm 3 . All shivers were digested at 37 °C in PBS solution containing 0.09% collagenase I (Sigma-Aldrich, St. Louis, MO, USA). Every 3-5 minutes, the digest was sucked into the prepared Dulbecco's modified Eagle's medium (DMEM)/F-12 (1:1) (Gibco by Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (Thermo Fisher Hyclone, Logan, UT, USA), and 100 U/ml penicillin -streptomycin to terminate digesting. About repeating 5-6 times, the shivers could be digested completely. The dissociated cells were collected by centrifugation at 118 × g for 6 minutes and suspended again in DMEM/F-12. Bromodeoxyuridine (BrdU, Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 0.1 mM was added during the first 36 hours to prevent proliferation of cardiac fibroblasts. Myocyte purity was about 95% 48 hours after plating. The cultures were maintained at 37 °C in a humidified 5% CO 2 atmosphere incubator. Experiments were performed on days of 3-5.
Reverse transcription-PCR (RT-PCR) experiment. Total RNA of cultured ventricular myocytes were isolated and cDNA was synthesized with the RevertAid TM FirstStrand cDNA Synthesis system for RT-PCR kits (Fermentas, Burlington, ON, Canada). Methods were similar to our previous report with slightly modified 36,37 . Primers used for RT-PCR analysis of all gene expressions were listed on Supplementary Table S1. The cycling parameters were as following: one cycle of 94 °C for 5 minutes and 35 cycles of 94 °C for 15 seconds, 47 °C for 45 seconds, 72 °C for 30 seconds followed by a single 10-minute cycle at 72 °C for extension. RT-PCR products were electrophoresed on a 2% agarose gel by using PCR markers (Tiangen Biotech, China) as the standard to determine the molecular size. Analysis was performed with GENIUS bioimaging system (Kodak, Rochest, NY, USA). Samples without the addition of reverse transcriptase or without the addition of RNA were as negative controls.
Immunofluorescence experiment. Ventricular myocytes were fixed with 4% paraformaldehyde in 0.01 M PBS, pH 7.4 for 30 minutes and then rinsed three times with PBS for 10 minutes. Followed by permeabilized with Figure 8. Schematic diagram of mechanisms for the extracellular acidification inducing "Ca 2+ transients" in cultured rat cardiac myocytes. There are two components for Ca 2+ elevation in response to elevated external protons, one is ASIC/TRPV1 channel and another is OGR1protein. On one side, ASICs and TRPV1 can be activated by acidosis solution and mediate extracellular Ca 2+ entry. On the other side, OGR1 mediates mobilization of intracellular Ca 2+ from SR via OGR1-PLC-IP3-IP3R signaling pathway.