Calcium Imaging in mouse adrenal glands. 70µm sections cut with 7000smz-2 vibrotome.

Abstract

(from: https://doi.org/10.1073/pnas.2014876118; "Enhanced Ca^2+ signaling, mild primary aldosteronism, and hypertension in a familial hyperaldosteronism mouse model (Cacna1h^M1560V/+)", Seidel et al, 2021)

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension

in humans. We used CRISPR/Cas9 to generate Cacna1h^M1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h^−/−). Adrenal morphology of both Cacna1hM1560V/+ and Cacna1h^−/− mice was normal. Cacna1h^M1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone

was only elevated in male animals. The systolic blood pressure of Cacna1h^M1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h^−/− mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a

cellular level, Cacna1h^M1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca^2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting.

Method

After removal of surrounding fat, both glands were embedded into 3% low-melting temperature agarose in BBS and glued to the mounting stage of a vibratome (7,000 smz-2; Campden Instruments). Organs in the agarose block were maintained in gassed BBS at temperatures below 4 °C at all times. Slices were cut at 70-μm thickness and transferred to BBS at 35 °C for 30 min for regeneration. Afterward, adrenal slices were stored for up to 6 h in BBS supplemented with 2 mmol/L CaCl2 at room temperature (∼22 °C).