Role of α2-Adrenoceptor Subtypes in Suppression of L-Type Ca2+ Current in Mouse Cardiac Myocytes

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca2+ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca2+ current (ICaL). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of ICaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of ICaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting

Store-operated Ca2+ entry supports contractile function in hearts of hibernators.

Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C-7°C-30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states

Ca²⁺ store-dependent regulation of contractile function of ground squirrel versus rat PM.

<p>(<b>A</b> and <b>B</b>) Representative recordings of ground squirrel and rat PM contractility under transition from nominal 1.8 mM Ca²⁺ to Ca²⁺-free Tyrode solution at 0.1 Hz stimulation frequency. (<b>C</b>) Same protocol in ground squirrel PM at doubled external Ca²⁺ levels also increase the force of contraction and further enhanced the amplitude of resting tension. Arrows in panels point to an increase in resting tension observed in ground squirrel but not rat PM. (<b>D</b> and <b>E</b>) Comparison of averaged relative force of contraction and relative resting tension (n = at least 3 in each experimental group) measured in the presence of CPA relative to control force of contraction before treatments. *, denotes statistically significant difference (P<0.05) defined by single group t-test comparing values with 1 in panel (<b>D</b>) and with 0 in panel (<b>E</b>). ‡, denotes significant difference (P<0.05) between relative resting tensions at 1.8 mM versus 3.6 mM of external Ca²⁺ in ground squirrel PM. (<b>F</b>) The profile of changes in the force of contraction and resting tension under manipulations with external Ca²⁺ and CPA was unaffected by the blocker of L-type Ca²⁺ channels, nifedipine, in line with moderate dependence of PM contractility on voltage-gated Ca²⁺ entry at low stimulation frequencies (<b>G</b>; *, P<0.05 for effects of nifedipine compared to corresponding controls; n = 4 in each group). (<b>H</b>) In ground squirrel PM, 2-APB reversed the contraction force potentiated by CPA to value below control. The averaged (n = 3) relative force of contraction is shown in panel (<b>D</b>).</p

Temperature-dependent adaptation of papillary muscle contractility in ground squirrel and rat hearts.

<p>(<b>A</b>) representative relative isometric contractile forces measured at 0.1 Hz stimulation frequency in isolated papillary muscles from ground squirrel (solid lines) and rat (dotted lines) hearts at different temperatures (as indicated). Contractile forces in both species were plotted relative to the values obtained at 30°C. (<b>B</b>) Summary statistics for the contractility of papillary muscle under conditions indicated in (<b>A</b>); *, denotes statistically significant differences (P<0.05, n shown in parenthesis) estimated using the single group t-test compared to maximal force of contraction before cooling (100%). (<b>C</b> and <b>D</b>) FFR constructed at 30°C before cooling to 10°C and after reheating; point values were obtained by normalizing measured maximal contraction forces to the averaged value at 0.1 Hz before cooling; *, denotes statistically significant differences between values before cooling and after reheating (P<0.05; n = 7 in ground squirrels, and n = 4 in rats).</p

CPA-induced changes in PRP and FFR of PM contractile function.

<p>(<b>A</b> and <b>B</b>) Representative recordings of ground squirrel and rat PM contractility implementing 2 s- and 60 s-long pauses along with corresponding PRP, calculated as a percentile ratio of the first post-rest contraction force <b>F</b>_<b>1</b> to the basal rhythmic contraction force <b>F</b>_<b>0</b> (at 1 Hz), plotted as a function of pause durations in the absence and presence of CPA. (<b>C</b>) and (<b>D</b>) Ground squirrels and rat FFR in the absence and presence of 10 μM CPA were constructed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177469#pone.0177469.g001" target="_blank">Fig 1C and 1D</a>. Insets at 0.1 and 1 Hz represent typical changes in PM contractility induced by CPA (dotted lines); *, denotes statistically significant CPA-induced changes in relative force contraction values (P<0.05; n = 9 in ground squirrels, and n = 3 in rats).</p

Changes of time-to-peak (TPT) and time to 50% relaxation (TP₅₀) contractile parameters induced by CPA (10 μM) in isolated PM.

<p>Changes of time-to-peak (TPT) and time to 50% relaxation (TP₅₀) contractile parameters induced by CPA (10 μM) in isolated PM.</p

2-APB- and CPA-induced modulation of ground squirrel PM contractility during cooling-reheating cycles.

<p>PM contractility acquired at 0.1Hz stimulation frequency in control (<b>A</b>), in the presence of 2-APB (<b>B</b>) or CPA (<b>C</b>). The temperature of bath solutions was changed at the approximate rate 0.1–0.2°C/min. Blue solid bars marked by asterisks (<b>B</b>) indicate 2-APB-induced inhibition of PM contractility before and after cooling, respectively. The profiles of temperature changes and drug application are shown by corresponding colored gradient bars and solid lines above the traces. (<b>D</b>) Summarized statistics for (<b>A</b>), (<b>B</b>) and (<b>C</b>) at the indicated temperatures; *, denotes statistically significant differences (P < 0.05; n = 5, 4 and 3 in control, in the presence of 2-APB and CPA groups, respectively), estimated using the single group t-test compared to peak force of contraction before cooling (100%).</p

Primer sequences for PCR amplification.

<p>Primer sequences for PCR amplification.</p

Effects of 2-APB on membrane currents in isolated cardiomyocytes and PM contractility in ground squirrel and rat hearts.

<p>(<b>A</b>) Representative voltage-current relationships of whole-cell membrane currents in a ground squirrel cardiomyocyte measured in response to a voltage ramp of 133.3 mV/s in the absence and presence of 2-APB. 2-APB-sensitive currents were obtained by subtraction of currents measured in the presence of 2-APB from control values at each membrane potential. (<b>B</b>) Time course and summary statistics of reversible 2-APB-induced inhibition of ground squirrel PM contractility measured at 0.1 Hz stimulation frequency. (<b>C</b>) FFR in ground squirrel PM in the absence (n = 8–9) and presence (n = 6) of 2-APB (20 μM); *, denotes statistically significant difference with P<0.05. (<b>D</b>) Representative voltage-current relationships of whole-cell membrane currents in a rat cardiomyocyte detected in response to a voltage ramp of 153.3 mV/s in the absence and presence of 2-APB. 2-APB-induced currents were obtained by subtraction of currents measured in the presence of 2-APB from control values at each membrane potential. (<b>E</b>) Representative recordings at 0.3 Hz stimulation frequency exemplifying the incidents of contractile dysfunction and arrhythmia induced by 2-APB in rat, but not in ground squirrel PM.</p