PMC

Constitutively active calcineurin rescues ventricular hypertrophy during chamber formation in the absence of L-type voltage-gated Ca²⁺ influx or contraction. Embryos were injected with (A) cacna1c morpholinos (MO) alone (n=19) or (B) co-injected with cacna1c MO and a cRNA rescue construct encoding a constitutively active calcineurin (caCN) (n=12). (C) Quantitation of outer curvature ventricular cardiomyocyte cell volume demonstrated that constitutively active calcineurin rescued cardiomyocyte growth during chamber formation in the absence of Ca_V1.2-mediated Ca²⁺ influx or contraction. Examples of representative measured cells are shaded in white. * P < 0.0001 vs cacna1c MO. Scale bar = 20 μM.

Three-dimensional volumetric analysis of ventricular cardiomyocyte hypertrophy during zebrafish chamber formation. (A) Linear heart tube at 24 hpf prior to chamber expansion (n=10). (B) Expanded ventricle at 48 hpf (n=8). (C) Quantitation of cell volumes demonstrated outer curvature ventricular cardiomyocytes increased 3- to 4-fold in volume during chamber formation primarily due to cytoplasmic expansion. Examples of representative measured cells are shaded in white. * P < 0.001 vs 24 hpf. Scale bar = 20 μM.

Contraction and blood flow are not required for ventricular hypertrophy during chamber formation. Zebrafish hearts were rendered non-contractile during chamber formation by treatment with blebbistatin or tnnt2 morpholino (MO). Of note, Ca²⁺ transients and electrical conduction are preserved with both treatments. No difference in outer curvature ventricular cardiomyocyte cell volume was found between hearts treated with (A) DMSO (n=8) and (B) blebbistatin (n=11) between 24 hpf and 48 hpf. Similarly, no difference in outer curvature cardiomyocyte cell volume was found between zebrafish embryos injected with (C) control MO (n=7) and (D) tnnt2 MO (n=6) when measured at 48 hpf. Examples of representative measured cells are shaded in white. Scale bar = 20 μM.

Increasing Ca_V1.2-mediated Ca²⁺ signaling during chamber formation leads to enhanced developmental hypertrophy. Voltage-gated Ca²⁺ entry via the Ca_V1.2 L-type Ca²⁺ channel was increased during development by expression of the Timothy Syndrome mutant Ca_V1.2 channel. Co-injection of cacna1c morpholinos (MO) with (D) Ca_V1.2^TS cRNA (TS, n=7) led to increased outer curvature ventricular cardiomyocyte cell volume at 48 hpf when compared to embryos injected with (A) control MO (n=7), (B) cacna1c MO alone (n=19), or (C) cacna1c MO with Ca_V1.2^WT cRNA (WT, n=8). Examples of representative measured cells are shaded in white. * P < 0.01 vs control MO. Scale bar = 20 μM.

Increasing Ca_V1.2-mediated Ca²⁺ signaling during chamber formation leads to enhanced developmental hypertrophy independent of contraction and blood flow. L-type voltage-gated Ca²⁺ entry was increased during development by treatment with the Ca_V1.2 agonist BayK8644 (BayK). Experiments were repeated in the absence of contraction by co-treatment with blebbistatin or tnnt2 morpholino (MO). Treatment of contractile hearts with (B) BayK (n=13) between 24 hpf and 48 hpf led to increased developmental hypertrophy when compared with (A) DMSO (n=8). The ability of BayK treatment to increase developmental hypertrophy was reproduced in hearts rendered non-contractile through treatment with (C, D) blebbistatin (n=11 for blebbistatin, n=8 for blebbistatin+BayK) or (E, F) tnnt2 MO (n=6 for tnnt2 MO, n=7 for tnnt2 MO+BayK). Examples of representative measured cells are shaded in white. * P < 0.05 vs paired control. Scale bar = 20 μM.

Calcineurin is required for ventricular hypertrophy independent of contraction and blood flow. Calcineurin was inhibited during development by treatment with cyclosporine A (CsA). Experiments were repeated in the absence of contraction by co-treatment with blebbistatin or tnnt2 morpholino (MO). Treatment of contractile hearts with (B) CsA (n=11) between 24 hpf and 48 hpf led to small ventricles with reduced cardiomyocyte cell volume when compared with (A) DMSO (n=8). Similar results were achieved in hearts rendered non-contractile during development through treatment with (C, D) blebbistatin (n= 11 for blebbistatin, n=12 for blebbistatin+CsA) or (E, F) tnnt2 MO (n=6 for tnnt2 MO, n=12 for tnnt2 MO+CsA). Examples of representative measured cells are shaded in white. * P < 0.01 vs paired control. Scale bar = 20 μM.

Ca²⁺ signaling via the Ca_V1.2 L-type Ca²⁺ channel is required for ventricular hypertrophy during chamber formation. L-type voltage-gated Ca²⁺ signaling was inhibited during chamber formation by treatment with nisoldipine or cacna1c morpholino (MO). Both treatments rendered hearts non-contractile during development in the absence of L-type voltage-gated Ca²⁺ transients. Ventricular chamber formation was grossly abnormal and outer curvature ventricular cardiomyocyte cell volume was significantly reduced in hearts treated with (B) nisoldipine (n=9) vs (A) DMSO (n=15) between 24 hpf and 48 hpf. More pronounced findings were observed in hearts injected with (D) cacna1c MO (n=19) vs (C) control MO (n=7) when measured at 48 hpf. Examples of representative measured cells are shaded in white. * P < 0.05 vs DMSO or control MO. Scale bar = 20 μM.