Initial characterization of a transgenic mouse with overexpression of the human D1-dopamine receptor in the heart

Fig. 1. Scheme. D1-dopamine receptors (D1R) are located in the sarcolemma of cardiomyocytes. They can be stimulated by dopamine or the D1R-agonist SKF38393. SCH22390 is an antagonist at D1R. When D1R have been activated, the activity of adenylyl cyclases (AC) is enhanced via GTP-binding stimulatory proteins (Gs). This leads to formation of cAMP and activation of cAMP-dependent protein kinases (PKA). PKA can activate by phosphorylation the L-type calcium ion channels (LTCC) or the ryanodine receptor (RYR) or phospholamban (PLB) or the troponin inhibitor (TnI)

Fig. 2. A Autoradiography. Representative images of autoradiography of D1-TG and WT mouse heart sections labeled with 5 nM [3H]SCH23390 (total binding, TB) or 5 nM [3H]SCH23390 + 10 M butaclamol (unspecific binding, UB). B In situ hybridization. Expression of D1-dopamine receptor mRNA detected by DIG-labelled human D1-dopamine receptor antisense probes (A-X) and DIG-labelled mouse D1-dopamine receptor antisense probes (a-l). DIG-labeled sense probes were used as controls. (A-H): Low magnification microscopic images, scale bar = 500 µm. mRNA expression of D1-dopamine receptor was not detected in WT mouse atria (A) and ventricles (C); whereas expression of human D1-dopamine receptor mRNA was detected in D1-Tg mouse atria (E) and ventricles including interventricular septum (G). (B, D, F, H) are corresponding control sections hybridized with DIG-labeled sense probes. (I-X): High magnification microscopic images, scale bar = 50 µm. No or very scarce and weak D1-dopamine mRNA expression was detected in WT mouse atria (I) and interventricular septum (K), while abundant human D1-dopamine mRNA expression was detected in atriums (M), interventricular septum (O), and ventricle walls (Q, S). (J, L,…

Fig. 3. Western blots. A A typical Western blot for the D1 receptor in hearts from WT, D1-TG, and D1-KO after incubation with the D1-dopamine receptor antibody is seen. The corresponding band for the D1 receptor (DRD1) is labelled with an arrow. As loading control, the Ponceau S-stained membrane is shown below. B Typical Western blots for regulatory proteins in hearts from WT and D1-TG after incubation with the D1-dopamine receptor agonist SKF 38393 are seen. Western blots depict the phosphorylation state of inhibitory subunit of troponin (P-TnI) with arrows. As a loading control, we assessed the protein expression of cardiac calsequestrin (CSQ) by cutting horizontally the lanes of the blot and incubating the lower and upper halves with different primary antibodies. M rainbow marker, LA left atrium, RA right atrium, VT ventricle

Fig. 4. Original recordings: contractile effects of dopamine. Isolated electrically stimulated left atrial preparations from WT (A) and D1-TG (B) were studied. Ordinates give force of contraction in millinewton (mN). Horizontal bar indicates time in minutes (min). First propranolol alone was given (10 µM). After 10 min of pre-treatment dopamine was cumulatively applied. Time points of addition of dopamine are indicated with arrows pointed upwards. Concentrations of dopamine are indicated as negative decadic logarithms. Dopamine increases force of contraction only in preparations from D1-TG. C The same experiments were carried out here as in (A) and (B), but finally a concentration-response-curve with the D1 receptor antagonist SCH23390 was added

Fig. 5. Contractile effects of dopamine in left atrial preparations from WT. Several experiments on isolated electrically stimulated left atrial preparations from WT such as in Fig. 4 are summarized here. Ordinates in (A) and (F) give force of contraction in millinewton (mN). Ordinates in (B) and (C) give rate of contraction and rate of relaxation in millinewton per second (mN/s). Ordinates in (D) and (E) give time to peak tension and time of relaxation in milliseconds (ms). Abscissae in this figure indicate first application of 10 µM propranolol (Pro) and subsequent concentrations of dopamine in negative decadic molar concentrations. * and # indicate significant differences versus control (Ctr; pre-drug value) or propranolol, respectively. “n” indicates number of experiments

Fig. 6. Contractile effects of dopamine in left atrial preparations from D1-TG. Several experiments on isolated electrically stimulated left atrial preparations from D1-TG such as in Figure 4 are summarized here. Ordinates in (A) and (F) give force of contraction in millinewton (mN). Ordinates in (B) and (C) give rate of contraction and rate of relaxation in millinewton per second (mN/s). Ordinates in (D) and (E) give time to peak tension and time of relaxation in milliseconds (ms). Abscissae in this figure indicate first application of 10 µM propranolol (Pro) and subsequent concentrations of dopamine in negative decadic molar concentrations. * and # indicate significant differences versus control (Ctr; pre-drug value) or propranolol, respectively. “n” indicates number of experiments

Fig. 7. Original recordings: effect of SKF 38393 on left atrial preparations in WT (A) and D1-TG (B). Isolated electrically stimulated left atrial preparations from WT (A) and D1-TG (B) were studied. Ordinates give force of contraction in millinewton (mN). Horizontal bar indicates time in minutes (min). First propranolol alone was given (10 µM). After 10 min of pre-treatment, SKF 38393 was cumulatively applied. Time points of addition of SKF 38393 are indicated with arrows pointed upwards. Concentrations of SKF 38393 are indicated as negative decadic logarithms. SKF 38393 increases force of contraction only in preparations from D1-TG

Fig. 8. Contractile effects of SKF 38393 in left atrial preparations from WT. Several experiments such as in Fig. 7A and, in addition, with a final concentration-response-curve with the D1 receptor antagonist SCH23390 are summarized here. Isolated electrically stimulated left atrial preparations from WT were studied. Ordinates in (A) and (F) give force of contraction in millinewton (mN). Ordinates in (B) and (C) give rate of contraction and rate of relaxation in millinewton per second (mN/s). Ordinates in (D) and (E) give time to peak tension and time of relaxation in milliseconds (ms). Abscissae in this figure indicate first application of 10 µM propranolol (Pro) and subsequent concentrations of SKF 38393 in negative decadic molar concentrations. * and # indicate significant differences versus control (Ctr; pre-drug value) or propranolol, respectively. “n” indicates number of experiments

Fig. 9. Contractile effects of SKF 38393 in left atrial preparations from D1-TG. Several experiments such as in Fig. 7B and, in addition, with a final concentration-response-curve with the D1 receptor antagonist SCH23390 are summarized here. Isolated electrically stimulated left atrial preparations from D1-TG were studied. Ordinates in (A) and (F) give force of contraction in millinewton (mN). Ordinates in (B) and (C) give rate of contraction and rate of relaxation in millinewton per second (mN/s). Ordinates in (D) and (E) give time to peak tension and time of relaxation in milliseconds (ms). Abscissae in Fig. 9 indicate first application of 10 µM propranolol (Pro) and subsequent concentrations of SKF 38393 in negative decadic molar concentrations. * and # indicate significant differences versus control (Ctr; pre-drug value) or propranolol, respectively. “n” indicates number of experiments

Fig. 10. Original recordings of the effects of dopamine in right atrial preparations from WT (A) and D1-TG (B, C). Isolated spontaneously beating right atrial preparations (RA) from WT (A) and D1-TG (B, G) were studied. Ordinates in (A) to (F) give beating rates in beats per minute (bpm). Ordinates in (G) give force of contraction in millinewton (mN). Horizontal bars indicate time in minutes (min) in (A), (B), and (G). First, propranolol alone was given (10 µM). After 10 min of pre-treatment, in addition dopamine was cumulatively applied. Finally, the D1 receptor antagonist SCH23390 was cumulatively applied. Time points of addition of dopamine or SCH23390 are indicated with arrows pointed upwards. Concentrations of dopamine in Fig. 10G are indicated as negative decadic logarithms. Dopamine increases beating rate and force in right atrial preparations from D1-TG. Abscissae in thid figure indicate first application of 10 µM propranolol (Pro) and subsequent concentrations of dopamine in negative decadic molar concentrations. * and # indicate significant differences versus control (Ctr; pre-drug value) or propranolol, respectively. “n” indicates number of experiments

Fig. 11. Original recordings of SKF 38393 in right atrial preparations from WT (A) and D1-TG (B, C). Isolated spontaneously beating right atrial preparations (RA) from WT (A) and D1-TG (B, C) were studied. Ordinates in (A), (B), and Fig. 10D give beating rates in beats per minute (bpm). Ordinate in (C) gives force of contraction in millinewton (mN). Horizontal bars indicate time in minutes (min) in (A), (B), and (C). First propranolol alone was given (10 µM). After 10 min of pre-treatment, in addition SKF 38393 was cumulatively applied. Time points of addition of SKF 38393 are indicated with arrows pointed upwards in (A), (B), and (C). Concentrations of SKF 38393 are indicated as negative decadic logarithms. SKF 38393 increases beating rate and force in right atrial preparations from D1-TG. In (F) and (G), the D1 receptor antagonist SCH23390 was cumulatively applied after SKF 38393. Abscissae in this figure indicate first application of 10 µM propranolol (Pro) and subsequent concentrations of SKF 38393 in negative decadic molar concentrations. * and # indicate significant differences versus control (Ctr; pre-drug value) or propranolol, respectively. “n” indicates number of experiments